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LIBRA R. Y

Michigan State
University

TH 8318

This is to certify that the

thesis entitled

Developmental Biology of Interclonal
Juniperus L. Grafts.

 

presented by

George Edward Evans

has been accepted towards fulfillment
of the requirements for

Ph.D.

degree i“Horticulture

 

 

 

0-169

l

ABSTRACT

DEVELOPMENTAL BIOLOGY OF INTERCLONAL
JUNIPERUS L. GRAFTS

By
George Edward Evans

Using several clones of Juniperus L., a series of experiments
were conducted to study various aspects of self and interclonal grafts
during their development.

Cytological studies produced new chromosome counts for three
previously uncounted juniper clones.

An anatomical study revealed an increase in medullary ray cell
size with increase in ploidy level.

In developing grafts, no basic difference was noted between clones
used, only differences in the time each stage was reached. The healing
sequence, beginning at 20 days, was as follows: filling of voids with
callus originating outside the xylem; overwalling of injured xylem
surfaces with isodiametric cells originating near the cambium; division
and differentiation of overwalling cells into typical vascular and
cambial cells; and cell production from the new cambial initials.
Abnormally large numbers of pits and occasional "blind pits" were
observed in mixed graft tissue.

Mineral distribution studies of entire graft components using
ashing and photometry techniques revealed that apparent rate of trans-
location at the graft union was in decreasing order: potassium, calcium,

and magnesium. Greatest accumulation of potassium occurred in Juniperus

chinensis 'Hetzii' scions and lowest in scions of Juniperus horizontalis

'Plumosa Compacta' self grafts.

2 George Edward Evans

Mineral distribution, using the electron microprobe X-ray analyzer,
showed translocation rates to be in decreasing order: magnesium, potas—
sium, and calcium. Greatest blockage of calcium appeared to be at the
xylem union, while potassium and magnesium moved relatively unimpeded.
In phloem and cortical tissue, calcium accumulated in the scion while
other elements were equally distributed between stock and scion.

Serological techniques, using fluorescein labelled antibody
obtained by immunizing inbred mice with juniper leaf and stem extract,
made possible the identification, in mixed graft tissue, of cells
belonging to each component of interclonal grafts. Although antibody
specificity was greatest in parenchymatous tissue, the greatest

fluorescence occurred in the xylem of Juniperus horizontalis 'Fountain'.

 

Cultural studies of self and interclonal grafts revealed greatest
success in 'Fountain' self grafts and those involving this clone as a
component, and lowest in self grafts and those involving 'Pfitzeriana
Kallay'.

Experiments in which the leaf—stem extract of one juniper was
applied to cut surfaces of self grafts, at grafting, showed that graft
success of 'Pfitzeriana Kallay' could be increased by application of
'Fountain' extract. When 'Pfitzeriana Kallay' extract was applied to

'Fountain' or Juniperus chinensis 'Hetzii' self grafts, success and

 

vigor was reduced.

Cell measurements revealed distinct differences in ray cell
dimensions and tracheid length among the clones. Tracheids formed
subsequent to graft wounding were larger than those in uninjured

tissue.

3 George Edward Evans

Cell counts indicated that generally greater numbers of tracheids
arose from understocks in typical grafts than from scions. In self
grafts greatest cell contributions occurred in graft components of
'Hetzii'. New tracheid production was delayed in 'Pfitzeriana Kallay'
self grafts until after 30 days, while a shorter delay occurred in
'Fountain' self grafts. Although occurring at different periods in
graft development, plateaus in new cell production were noted in
'Hetzii' self grafts.

Scion cell production increased significantly with increasing
ploidy level of the understock except where 'Pftizeriana Kallay' was
the scion. 'Hetzii' scions and ‘Pfitzeriana Kallay' understocks
produced greatest amounts of new cells in uninjured wood, while in
wound tissue more cells arose from 'Pfitzeriana Kallay' scions.

Histochemical studies using Ruthenium Red staining showed pectic
substances to be concentrated in the middle lamella of mature tracheids
but very diffuse in undifferentiated tissue.

In addition to increasing our basic knowledge of the graft healing
sequence of certain Juniperus L. clones, the present study brought to

light new avenues of approach to graft union study.

 

DEVELOPMENTAL BIOLOGY OF
INTERCLONAL JUNIPERUS L. CRAFTS

By

George Edward Evans

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Horticulture

1969

 

 

56277:?
7— /’ 7 0

ACKNOWLEDGMENTS

The author wishes to extend his sincere appreciation to his
major professor, Dr. H. P. Rasmussen, for his special assistance and
valuable suggestions during completion of degree requirements and to
his guidance committee, Drs. Davidson, Pollard, Mecklenburg, and
Hooper, for their constructive suggestions during preparation of the
dissertation. A special note of appreciation is extended to the
author's wife, Barbara Jeanne, for her assistance, encouragement, and

patience during the extended period of degree requirement completion.

ii

TABLE OF CONTENTS

Page Number

Section I ”Chromosome Counts In Three Cultivars

of Juniperus L. " . . . . . . . . 1
Introduction . . . . . . . . . 1
Materials and Methods. . . . . . . . . . . . . 3
Results and Discussion . . . . . . . . . . . . . . . 3
Summary. . . . 9
Literature Cited 10

Section II "Anatomical Changes in Developing Graft
Unions of Juniperus L. ". . . . . . . . . . . . 11
Introduction . . . . . . . . . . . . . . . 11
Materials and Methods. . . . . . . . . . . . . . . . . . 14
Results and Discussion . . . . . . . . . . . . . . . . . 17
Summary. . . . . . . . . . . . . . . . . . . . . . . 29
Literature Cited . . . . . . . . . . . . . . . . . . . . 31
Section III ”Distribution of Calcium, Potassium, and
Magnesium in Interclonal Grafts of
Juniperus L.”. . . . . . . . . . . . . . . . . 33
Introduction . . . . . . . . . . . . . . . . . . . 33
Materials and Methods. . . . . . . . . . . . . . . . . . 36
Results and Discussion . . . . . . . . . . . . . . . . . 39
Summary. . . . . . . . . . . . . . . . . . . . . . . 49
Literature Cited . . . . . . . . . . . . . . . . . . . . 52
Section IV "Serological Determination of Tissue
Contribution from Stock and Scion in

Juniperus L. Grafts”. . . . . . . . . . . . . . 54
Introduction . . . . . . . . . . . . . . . . . 54
Materials and Methods. . . . . . . . . . . . . . . . . . 56
Results and Discussion . . . . . . . . . . . . . . . . . 62
Summary. . . . . . . . . . . . . . . . . . . . . . 73
Literature Cited . . . . . . . . . . . . . . . . . . . . 76

 

 

Page Number

Appendix A "Cytological, Cultural, and Histological
Studies of the Progressive Development of

Interclonal Juniperus L. Crafts" 78
Introduction. . . . . . . . . . . 78
Materials and Methods . . . . . . . 92
Results and Discussion. . . 96
Summary . . . . . . . . . . . . . . . . . . . . . . 118
Literature Cited. . . . . . . . . . . . . . . . . . . 122

Dissertation
Summary . . . . . . . . . . . . . . . . . . . 127
Appendix B . . . . . . . . . . . . . . . . . . . . . . . . . 132

iv

 

Section I
Table 1

Section II
Table 1

Section III

 

Table 1

Table 2

Table 3

Appendix A
Table 1

Table 2

Table 3

Table 4
Table 5

Table 6

Table 7

LIST OF TABLES

A compilation of documented chromosome
counts for Juniperus L.

Histological observations on Juniperus L.
grafts at six dates following grafting .

Stock-scion ratios for calcium content of
juniper graft combinations sampled at
regular intervals after grafting . .
Stock-scion ratios for potassium content

of juniper graft combinations sampled at
regular intervals after grafting . . . . .
Stock-scion ratios for magnesium content of
juniper graft combinations sampled at
regular intervals after grafting .

"Intergeneric" hybrids in the Rosaceous
subfamily Pomoideae Focke. . . . . .
Results of grafting experiments in the
genus Viola (from Dodd and Gershoy,

1943) . . . . . . . .
Survival and average shoot elongation of

4 month old grafts (from Evans, Watson,
and Davidson, 1961).. . . . . . . .
Surviving juniper Stock/scion combinations
on July 27,1959 (from Van Sloun, 1959).
Craft survival for six interspecific pine
graft combinations (from Ahlgren, 1962).
Survival counts of 5 month old juniper
grafts based on 12 plants per

combination. .
Survival counts and visual observations of
5 month old tissue extract treated self
grafts based on 5 plants per

combination.

Page Number

 

19

41

42

43

79

8O

82

83

84

98

102

 

 

Appendix A
Table 8

Table 9

Table 10

Table 11

Appendix B
Table B1

Table B2

Table B3

Table B4

Mean tracheid and ray cell measurements
(n=5), coefficients of variability, and
Duncan's multiple range scores for data
from two-year— old stems. .
Radial cell count and stock-scion means
for un—wounded xylem in interclonal
Juniperus L. graft partners at 5
postgraft dates. .
Radial cell count and stock— —scion means
for wounded xylem in interclonal
Juniperus L. graft partners at 5
postgraft dates.

Comparative mean radial cell counts for.
wound induced and normal tissue produced
after grafting in nine interclonal
juniper graft combinations

Radial cell counts
tissue produced by
juniper grafts and
5 dates subsequent
Radial cell counts
tissue produced by
juniper grafts and
5 dates subsequent
Calcium, magnesium

of normal xylem
various interclonal
their components at
to grafting .

of wound xylem
various interclonal
their components at
to grafting

and potassium content

of juniper grafts and their component
tissues harvested at three dates
subsequent to grafting .

Analyses of variance for stock—scion

mineral content ratios of juniper grafts

sampled at regular
grafting . . . . .

intervals after

vi

Page Number

105

108

109

114

132

133

134

135

 

 

Section I
Figure 1

Figure 2

Figure 3

Section II
Figure 1

Figure 2

Figure 3

Section III

 

Figure 1

Figure 2

Section IV
Figure 1

Figure 2

LIST OF FIGURES

Page Number

 

Photomicrograph of the triploid (3n=33)

chromosome complement of Juniperus

chinensis 'Hetzii' . . . . . 4
Photomicrograph of the diploid (2n=22)

chromosome complement of Juniperus

horizontalis 'Plumosa Compacta'. . . . . 5
Photomicrograph of the diploid (2n=22)

chromosome complement of Juniperus

sabina 'Von Ehron' . . . . . . . . . . . . . 6

 

Progressive histological change of
juniper grafts during the period

zero to 60 days after grafting . . . . . . . 20
Fine structure and origin of graft

union tissues. . . . . . . . 25
Pitting behavior among mixed vascular

cells of stock and scion . . . . . . . . . . 27

Line profile analysis of a juniper

graft union showing A) distribution

K, Mg and Ca; and B) secondary electron

oscillogram with line scan from 1 to 2 . . . 46
Mean calcium, potassium, and magnesium

content of interclonal juniper graft

tissues within stock and scion as

measured by electron microprobe

X—ray analysis . . . . . . . . . . . . . . . 48

Microprecipitin test plates for antisera
of Juniperus horizontalis 'Fountain'
(A) and Juniperus chinensis 'Pfitzeriana

 

 

Kallay' (B). . . . . . . . 64
Background fluorescence of non—fluorescein
labelled xylem and cortex. . . . . . . . . . 65

vii

 

Section IV
Figure 3

Figure 4

Appendix A
Figure 1

Figure 2

Figure 3

Figure 4

Photomicrographs of fluorescent
antibody labelled stocks and scions

of Juniperus horizontalis 'Fountain'

 

on Juniperus chinensis

Kallay' grafts .

Fluorescent antibody labelling of

graft unions

Diagramatic representation of a
typical 60 day old graft union viewed in
Points are indicated at

cross-section.

which cell counts and cell measurements

were taken .

The effect of application of leaf and
stem extract to graft surfaces of
Juniperus chinensis 'Pfitzeriana

O

 

Kallay'

grafting .

Pectic substance localization in juniper
graft unions and in medullary ray cell

walls.

self grafts. .
Mean cell counts at wounded and un-wounded
sites of stocks and scions of interclonal
juniper grafts at five dates after

viii

O

'Pfitzeriana

Page Number

 

68

71

9'7

101

111

117

 

 

Guidance Committee:

Sections I, II, III, and IV are segments of
related thesis research information condensed into formats suited,
and intended for publication in The Botanical Gazette, The
American Journal of Botany, Journal of the American Society for

Horticultural Science, and The Botanical Gazette, respectively.

ix

 

SECTION I

Chromosome Counts In Three Cultivars of Juniperus L.

Introduction

The first cytological studies within Juniperus L. were conducted
less than four decades ago (12). Considering the size of this genus,
it is surprising that documented chromosome counts exist for only 11
different species.

Early studies indicated Juniperus L. to have a base chromosome
number of nfill; one found to be consistant among other genera of the
Cupressaceae, but differing by one chromosome from what is believed
to be the original base number of n=12 for gymnosperms (12).

A compilation of documented chromosome numbers for Juniperus L.
is given in Table 1. This includes the counts reported by Darlington
and Wylie (2) as well as the Index to Plant Chromosome Numbers (1).
A11 counts listed as haploid resulted from gametophyte counts; the
remainder being from mitotic counts of root tips or seed endosperm.

Polyploidy is considered rare in Juniperus L., the only documented
counts being those reported for Juniperus chinensis and its cultivar
'Pfitzeriana' which were considered to be autotetraploids with 2n=44
(12). Incidental to a study of tetraploidy in Seguoiadendrom giganteum,
Jensen and Levan (4) reported a chromosome number (unpublished) of
2n=44 for Juniperus sguamata 'Meyeri'. A single case of triploidy in
juniper has been reported by Stiff in 1951 (13). This juniper was
discovered among open pollinated seedlings of Juniperus Virginiana
and had a chromosome count of 3n=33. No postulate was advanced by

the author concerning the possible pollen parent of this triploid.

Table l. A compilation of documented chromosome counts for Juniperus L.

 

 

Chromosome

Species Count .Contributor
Juniperus bermudiana n=11 Mehra and Khoshoo (9)
Juniperus chinensis 2n=44 Sax and Sax (12)
Juniperus communis 2n=22 Sax and Sax (12)
Juniperus communis 2n=22 Love and Love (8)
Juniperus communis 2n=22 Jorgensen and S¢rensen (6)

v. montana

Juniperus horizontalis 2n=22 Ross and Duncan (11)
Juniperus phoenicea n=11 Mehra and Khoshoo (9)
Juniperus procera 2n=22 Mehra and Khoshoo (9)
Juniperus rigida 2n=22 Sax and Sax (12)
Juniperus sabina 2n=22—24 Reese (10)
Juniperus Virginiana 2n=22,33 Stiff (13)
Juniperus Virginiana n=11 Mehra and Khoshoo (9)

v. scopulorum

 

3

Materials and Methods

The plant material employed in this study was obtained as clonally
propagated stock from a commercial nurseryl/. The cultivars chosen for
study were Juniperus chinensis 'Hetzii', Juniperus horizontalis
'Plumosa Compacta', and Juniperus sabina 'Von Ehron'.

Root tips 2—3 mm. in length were collected and placed in several
pretreatment solutions to determine a suitable one for arresting chromo—
somes at metaphase. Test solutions included 0.002 M 8—hydroxyquinoline
(14) or 0.2% aqueous colchicine for 4 or 6 hours. After pretreatment
the root tips were killed and fixed in modified Carnoy's (5) solution
(absolute ethanol, chloroform, glacial acetic acid, 6:3:1 v/v) prior to
cytological examination.

Fixed root tips were treated with 50% HCl for 10 minutes to
facilitate tissue maceration. The tissue was smeared on a microslide
and stained with 5% aceto—carmine and the coverslip temporarily sealed
with petroleum jelly. Sufficient numbers of preparations were examined
to obtain accurate chromosome counts. Photomicrographs were taken for

a permanent record of the observations.
Results and Discussion

Eight—hydroxyquinoline appeared to provide the best chromosome
preparations of the two pretreatments used. A 4 hour pretreatment

was sufficient for Juniperus chinensis 'Hetzii' while 6 hour

1] Monrovia Nurseries, Azusa, California

 

    

 

Figure l - Photomicrograph of the triploid (3n=33) chromosome
complement of Juniperus chinensis 'Hetzii'. Root
tips pretreated 4 hours with colchicine. X 1000.

 

 

 

Figure 2 —

 

Photomicrograph and interpretive drawing of the
diploid (2n=22) complement of Juniperus horizontalis
'Plumosa Compacta'. Root tips pretreated 4 hours
with 8-hydroxyquinoline. X 1900.

 

 

 

Figure 3 — Photomicrograph and interpretive drawing of the
diploid (2n=22) complement of Juniperus sabina
'Von Ehron'. Root tips pretreated for 6 hours
with 8-hydroxyquinoline. X 2100.

7

pretreatment resulted in better preparations for Juniperus horizontalis

 

'Plumosa Compacta' and Juniperus sabina 'Von Ehron'.

0f greatest interest was the observation of a triploid chromosome
count for Juniperus chinensis 'Hetzii'. On the basis of the known
tetraploid count for Juniperus chinensis (12), one would also expect
the cultivar 'Hetzii' to be 2n=44. One of the 10 smears clearly showing
the 3n=33 chromosome complement is presented in Figure 1.

Since this cultivar has been solely propagated by vegetative means
since its discovery, the unbalanced chromosome complement has been
maintained. The parentage of Juniperus chinensis 'Hetzii' is, at the
present, a matter of conjecture. According to Den Ouden (3), it was
obtained by Fairview Evergreen Nurseries, Fairview, Pa. in 1920.
However, it was suggested by Leiss (7) that this variant seedling was
obtained from the West coast, from a cross between Juniperus Virginiana
glauca as seed parent, and Juniperus chinensis 'Pfitzeriana' as pollen
parent.

During the course of this study, a diploid count of 2n=22 was

obtained for Juniperus horizontalis 'Plumosa Compacta' as shown in the

 

photomicrograph of Figure 2. It was possible to clearly obtain the
diploid count in the 15 individual cell smears found sufficiently well
prepared for accurate counting. This count was in agreement with the
diploid 2n=22 count reported for the species by Ross and Duncan (11).
Cytological studies of Juniperus sabina 'Von Ehron', consistantly
yielded a chromosome count of 2n=22 in 12 individual cell smears found
suitable for counting. The count is illustrated by the photomicrograph

of Figure 3, and supports the 2n=22—24 count reported for Juniperus

 

 

 

Summary

Somatic chromosome counts, by examination of root tip cells
were established for three cultivars of the genus Juniperus L..
A triploid mitotic count of 3n=33 was obtained for Juniperus chinensis
'Hetzii'. This was unexpected since this species has a previous
documented count of 2n=44 (12).

The 2n=22 obtained for Juniperus sabina 'Von Ehron' is in
accord with an earlier report of 2n=22—24 for the species (10). It
is quite possible that unbalanced genomes for junipers could survive
since they are propagated exclusively by vegetative means.

A somatic chromosome count of 2n=22 was found for Juniperus

horizontalis 'Plumosa Compacta'.

 

10.

11.

12.

13.

14.

10

Literature Cited

Cave, M. S. (editor). 1958-1964. Index to plant chromosome
numbers. Univ. North Carolina Press, Chapel Hill.

Darlington, C. D. and A. P. wylie. 1955. Chromosome atlas of
flowering plants. Ed. 2, London: Geo. Allen and Unwin Ltd.

Den Ouden P. and B. K. Boom. 1965. Manual of cultivated
conifers. The Hague, Netherlands, Martinus Nijhoff.

Jensen, H. and A. Levan. 1941. Colchicine induced tetraploidy
in Sequoiadendron gigapteum. Hereditas 27:220—224.

 

Johansen, D. A. 1940. Plant microtechnique. McGraw—Hill,
New York.

Jorgensen, C. D., S¢rensen, Th. and M. Westergaard. 1958. The
flowering plants of Greenland. A taxonomical and cytological
survey. Biol. Skr. Dansk. Vidensk. 9:1-172.

Leiss, J. 1966. Trials with three Juniperus understocks.
Proc. International Plant Prop. Soc. 16:215-217.

Love, A. and D. Love. 1956. Cytotaxonomical conspectus of the
Icelandic flora. Acta Horti Gotob. 20:65-290.

Mehra, P. N. and T. N. Khoshoo. 1956. Cytology of conifers I.
J. Genet. 54:165-180.

Reese, G. 1952. Ber. Dtsh. Bot. Ges. 65:241. As seen in:
Darlington, C. D. and A. P. Wylie. 1955. Chromosome atlas
of flowering plants. Ed. 2 London: Geo. Allen Unwin Ltd.

Ross, J. G. and R. E. Duncan. 1949. Cytological evidence of
hybridization between Juniperus Virginiana and Juniperus
horizontalis. Bull. Torrey Bot. Club 76:414-429.

 

Sax, K. and H. J. Sax. 1933. Chromosome numbers and morphology
in the conifers. J. Arn. Arb. 14:356-375.

Stiff, M. L. 1951. A naturally occuring triploid juniper.
Virginia J. Sci. 2:317.

Tjio, J. H. and A. Levan. 1950. The use of oxyquinoline in
chromosome analysis. Anales de la estacion experimental de
Aula dei 2:21—64.

SECTION II

 

11

Anatomical Changes in Developing Graft Unions of Juniperus L.

Until the advent of the electron microscope and radioactive
tracers, most graft studies were made at the morphological or gross
anatomical levels. Histological studies, however, date back to Waugh's
(1904) investigation of graft union callus tissue.

Although graft callus tissue has received much study, questions
concerning the relative proportions of callus produced by the scion,
the point of origin of callus tissue cells and its ultimate function
remain the object of controversy.

Aside from the function of wound protection, it has been suggested
(Sharples and Gunnery, 1933) that callus may serve (1) as temporary
nutrient transport from stock to scion, and (2) as a storage and
nutrient supply for regenerating vascular tissue. The reality of the
first listed function is demonstrated by Muzik's (1958) studies which
showed that vanilla grafts could survive and grow for at least 2 years
on parenchyma cell unions. This suggests the capability for both
acropetal and basipetal transport through these cells. Braun (1961)
credited callus tissue with the function of water supply to the scion
until a permanent vascular bridge was formed.

Kac (1931) believed that scions produced the greater amount of
callus tissue, while other workers (Kostoff, 1928; Mergen, 1955)
concluded that it was of stock origin. Sharples and Gunnery (1933),
on the other hand, claimed equal contributions from stock and scion.
Because of its more intact nature and greater food reserves, the stock

would logically be in better position to contribute to callus formation.

 

 

12
This would especially be true for conifers grafted with techniques in
which the entire stock is left intact until healing is complete.

Although callus would be expected to develop from any parenchyma—
tous cell near the graft union, it is generally agreed that it arises
from tissue outside the cambial ring in grafts of woody perennial
plants.

Apple graft callus has been described by Sass (1932) as arising
from tissue external to the xylem cylinder, exclusive of the periderm.
Other investigators (Sharples and Gunnery, 1933; Juliano, 1941) felt
that tissue within the xylem cylinder also contributed to callus
production. In cleft grafts of Nothopanax sp., Juliano (1941) found
callus to be first produced in the cortex, pith and in ray cells.
Medullary rays of the xylem were found to produce at least a portion of
the callus pad formed on xylem surfaces exposed by stripping the bark
of Hibiscus rosa—sinensis L. (Sharples and Gunnery, 1933). This
suggests that callus may arise from recently produced secondary xylem
and secondary phloem in the immediate vicinity of the union.

Within recent years, techniques have been perfected by which
callus tissues may be dissected from various plant parts and cultured
in vitro. Studies by Barker (1953), using tissue cultured from Tilia
americana, showed that medullary ray cells had the capacity to form
callus. This tends to support the hypothesis that the xylem may
contribute callus in woody plant grafts.

White's (1964, 1967) studies with Pipe§_glauca explants, supported

the theory that callus developed from the cambium, as well as the new

phloem. In old cultures, callus occasionally arose from xylem resin

 

l3
duct lining, but not from medullary ray tissue.

Although some doubt exists about the origin of cambial bridges
between stock and scion, certain authors (Sass, 1932; Mosse, 1962)
consider new cambial cells to arise through differentiation of callus;
a phenomenon also observed by Mosse and Labern (1960) in apple bud
unions and by Mathes (1967) in cultured explants of maple.

A chicory scion grafted into a root piece with mismatched cambiums
induced the formation, by dedifferentiation, and redifferentiation, of
a cribro—vascular system which connected existing vascular systems of
stock and scion (Games, 1949). This unexplained phenomenon was also
illustrated by Kuroda (1960a and 1960b) utilizing tissue culture grafts
of carrot. In this case, a scion containing xylem and phloem tissue
was grafted to stock tissue containing only phloem; the scion subse—
quently inducing formation of xylem elements in the stock. A review
by Gautheret (1966) suggested that by using tissue culture procedures
and adequate growth promoting substances, it should be possible for
virtually any cell type, including collenchyma, schlerenchyma, and
degenerative cells, to dedifferentiate.

Kostoff (1928), in studying callus, pointed to the possibility
that newly formed cambial initials could arise through radial divisions
of the cambial initials at the original point of incision. Kac (1931)
also suggested that new cambial derivatives pushed their way through
the wound callus.

To date, much has been learned about the general histology of the

graft union, but many questions are unresolved as to the nature of

common walls of mixed stock and scion cells. In intact homogeneous

 

14
cells, pit fields and subsequently formed pits appear to function as
sites of transport between cells. In a graft union, where each cell
is under different genetic control, these structures may pair at random.
If so, this may explain reduced water and mineral element transport
across less compatible grafts.

The pit field distribution in parenchyma cells of Aygpa coleop—
tiles, were found to remain constant in number and distribution during
cell extension (Wardrop, 1955). Wilson (1957) found that in mature
Elodea canadensis internodes longer than 0.5 mm. pit field behavior in
the primary cell walls, was similar to that in Aygpa coleoptiles
described by Wardrop. Applying these findings to graft union formation
may be difficult since each of the heterogeneous adjoining cells would
have to redistribute and pair pit fields following their formation
during cell division. Unless chance matching of pit fields had occurred,
translocation between cells would be greatly reduced, or limited to

diffusion through membranes.
Material and Methods

The genetically stable clonal forms Juniperus horizontalis
'Fountain', Juniperus chinensis 'Hetzii', and Juniperus chinensis
'Pfitzeriana Kallay' were used in this study and are referred to
hereafter by their cultivar names. Ploidy levels of these junipers
are 2n, 3n, and 4n respectively (Evans, 1969).

On October 24, 1966, the above listed junipers were obtained from

a commercial nursery£/, and after suitable cold treatment, potted in

if D. Hill Nursery Co., Dundee, Illinois.

 

15
4 inch clay pots and benched in a 68—700 F. night temperature green—
house. Scion wood, from the same nursery source, was stored at 350 F.
to break its rest period and assure continued dormancy. Stock and
scion wood were approximately one year old from cuttings; stem
diameters being l/8 to 3/16 inches.
Between January 15 and January 17, 1967, thirty each of the

following listed graft combinations were prepared using a modified

side graft:

Stock Scion
1. J. horizontalis 'Fountain' J. horizontalis 'Fountain'
2. J. chinensis 'Hetzii' J. chinensis 'Hetzii'

3. J. chinensis 'Pfitzeriana Kallay' J. chinensis 'Pfitzeriana Kallay'

4. J. chinensis 'Pfitzeriana Kallay' J. chinensis 'Hetzii'

5. J. chinensis 'Hetzii' J. chinensis 'Pfitzeriana Kallay'
6. J. horizontalis 'Fountain' J. chinensis 'Hetzii'

7. J. chinensis 'Hetzii' J. horizontalis 'Fountain'

8. J. horizontalis 'Fountain' J. chinensis 'Pfitzeriana Kallay'

9. J. chinensis 'Pfitzeriana Kallay' J. horizontalis 'Fountain'

The grafted plants were arranged in a randomized complete block
design incorporating 3 complete blocks; each treatment within the block
being replicated 10 times. All graft unions were covered with damp
Sphagnum moss, and the entire bench covered with 4 mil clear polyethy—
lene supported on a wooden frame 24 inches above the plants.

The understock plants were cut back to one—half their mass 40

days after grafting; the remainder being removed at the union 60 days

 

16
after grafting.

Progressive anatomical changes during healing were studied in stem
segments containing the graft union and collected at random from the
original grafted plants. Three samples of each combination were
collected at 10 day intervals throughout the experiment. Upon harvest
the samples were immediately frozen in polyethylene bags to await
sectioning.

Graft samples were evacuated and infiltrated for one hour in a
1.5% solution of Reteng/ using a venturi type vaccuum aspirator.
Sections were cut at 10 microns on a Model CTD refrigerated microtome
(cryostat)§/ at -150C.. Sections were picked up directly from the
microtome blade using microslides which had been previously coated with
ARG adhesive (Jensen, 1962) and a thin film of 0.75% Reten.

Sections were stained with safranin (15 minutes) and counter—
stained with aniline blue (1 minute): a stain combination considered to
be ideal for gymnosperms (Jensen, 1962).

The staining procedure was as described by Jensen (1962) but
included a stepped alcohol dehydration series between safranin and
aniline blue. Otherwise, poor preparations resulted when the sections
were placed in clove oil—absolute alcohol—xylene solution. The cover

4/

slips were mounted with Permount—-.

2] A cationic, water soluble polymer from Hercules Powder Co.
é] International Equipment Company

3] Fisher Scientific Company

17

Results and Discussion

General Observations

The ray cell size in each juniper clone studied was markedly
different. The tangential ray cell diameter differed considerably
from clone to clone, and was positively correlated with increasing
ploidy level: 'Fountain', 'Hetzii' and 'Pfitzeriana Kallay' (Evans,
1969). Differences in ray cell size in longitudinal section of a 40
day old graft of 'Fountain' (2n=22) on 'Pfitzeriana Kallay' (4n=44),
arrows indicating ray cells on either side of mixed graft union tissue,

are visible in Figure 1A.

Developmental Graft Histology

 

Typical progressive histological changes which occur at the graft
union during the healing period are presented in Figure l. Photomicro—
graphs were prepared which illustrate how the typical graft union
appeared at each of five dates following grafting. Figure 1 (A through
H) demonstrates the events noted during the period of zero to sixty
days after grafting. Observations of tissue production for all combina—
tions and dates are given in Table 1, and serve to compare the inter—
clonal grafts with respect to graft development.

Typical 10 day old grafts were characterized by a lack of activity
in most combinations; the most notable exception being the well formed
callus in 'Fountain' on 'Hetzii' combinations. The reciprocal of this
combination and 'Hetzii' on 'Pfitzeriana Kallay' produced moderate
amounts of callus tissue during the first 10 days.

Callus tissue was noted to arise rapidly from phloem and cortical

parenchyma during the initial stages of healing (Figure 1B and C) , an

 

l8
observation documented by other workers (Sass, 1932; Juliano, 1941;
Buck, 1954).

Callus tissue in 20 day old grafts had partially filled the void
between cut surfaces (at arrows, Figure 1B) with contributions coming
from both flanks of the graft incision. A limited amount of new tissue
had begun to form (Figure 1C, lower right) among the callus cells.
Mature tissue appears to be vertically bisecting this longitudinal
section. Such isolated tissue may have become separated from one of
the graft partners during grafting. The greatest contribution to new
xylem came from the understocks of 'Fountain' and 'Hetzii' self grafts,
whereas the interclonal grafts produced only a limited number of new
tracheids. Grafts involving 'Pfitzeriana Kallay' as a scion had not
produced new xylem by 20 days, and only small contributions of callus
from the scion had occurred at this date.

Immediately following or coincident with development of callus
tissue, certain cells, hereafter referred to as new xylem, were
observed to arise directly from the uninjured cambial surface. This
substantiates the observations of Sharples and Gunnery (1933) on
wound studies of Hibiscus rosa-sinensis, but is not in agreement with
the views of other workers (Sass, 1932; Mosse, 1962). A portion of
this tissue may arise directly from medullary rays as illustrated at
the arrows in Figure 2A. Such an occurrence has been reported by

Barker (1953) in tissue cultures of Tilia americana, and by White

 

(1964, 1967) in explants of Picea glauca. These cells were isodia—

 

metric (Figure 1G & 2A).

By an alternating series of radial and tangential cell divisions,

 

19

 

 

 

 

 

 

 

 

 

 

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20

Figure l - Progressive histological change of juniper grafts during
the period zero to 60 days after grafting.
(A) 'Fountain'/'Pfitzeriana Kallay', 40 days old, longitudinal
section (X 413). Mixed new vascular elements illustrating
distinguishable difference in ray cell size between clones.
(B) 'Fountain'/'Fountain', cross-section, 20 days old (X 41).
Beginning callus formation (at arrows) from phloem and cortex.
(C) 'Fountain'/'Fountain', longitudinal section, 20 days old
(X 100). New vascular elements forming at mature xylem—callus
junction (lower right). (D) 'Pfitzeriana Kallay'/'Fountain',‘
cross-section, 30 days old (X 30). Callus tissue continuing
to fill voids. (E) 'Fountain'/'Pfitzeriana Kallay' 40 days
old, cross-section (X 25). New vascular tissue (nxy) forming
from stock and scion. Largest increment from stock (right).
(F) 'Fountain'/'Hetzii', 50 days old, cross-section (X 165).
Contribution to new xylem (nxy) from stock and scion. Callus
(ca) crushed by advancing vascular elements. (G) 'Fountain'/
'Fountain', 40 days old, longitudinal section (X 83). Typical
view of vascular bridge with undifferentiated isodiametric
cells (arrow). (H) 'Fountain'/'Fountain',60 days old, cross-
section (X 35). Typical view of vascular bridge at later
stage of development. (I) 'Hetzii'/'Pfitzeriana Kallay', 60
days old, cross—section (X 165). Cambial bridging of union
and subsequent formation of normally oriented new xylem (nxy).

 

22

the wound between graft incisions became filled by new xylem which
crushed the callus tissue in its path (Figure 2B). Production of such
cambial derivitives continued by tangential division from each end of
the graft incision (Figure 2C), until all injured tissue was overlain
with new xylem.

Callus tissue had nearly filled all voids between graft partners
at 30 days except in 'Pfitzeriana Kallay' self grafts (Table 1).
Thirty day grafts were more sound and maintained their structural
integrity during sectioning. Graft incisions were, however, still well
defined, not having been obscured by newly formed xylem. At this date
most new xylem was still contributed primarily by the stock but the
scions showed increased cell production. No xylem production was noted
for 'Pfitzeriana Kallay' self grafts or grafts of this clone on
'Fountain' understocks.

Callus tissue was well developed in all graft combinations by the
40th day, and contribution to new xylem was predominately from the
stock (greater increment, Figure 1E). Callus and new xylem production
in 'Pfitzeriana Kallay' self grafts and in 'Pfitzeriana Kallay' on
'Fountain' grafts was observed for the first time at 40 days. An
example of such tissue can be seen in Figure 1E extending from the
flanks of the incision into the void between stock and scion. As a
result the intervening callus tissue was compressed into a darkly
stained mass of tissue (Figure 1F). Mosse (1962) also reported darkly
stained crushed cells between the tissues of stock and scion of fruit
bud unions.

At this point juniper graft development resembled closely that

23
found in wound healing of woody plants, especially in poorly matched
grafts. Bradford and Sitton (1929) drew a parallel between graft
healing and wound healing in such mismatched grafts. In closely fitted
grafts, production of new xylem between graft partners was considerably
reduced.

At 50 days the contribution to new xylem tissue was balanced
between stock and scion (Table 1). The tendency for 'Pftizeriana
Kallay' self grafts to produce limited xylem, was still evident. Con—
comitantly grafts involving this clone as a scion, heretofore lagging
in xylem production, produced moderate amounts.

At points along the graft incision where the stock and scion
cambia were close newly formed tissue became mixed to form "bridges"
(Figure 1G and 1H). Cambial initials within vascular bridges began by
tangential cell division to form normally oriented tracheids. The
mixed tissue lost the isodiametric shape of earlier formed cells and
assumed the typical linear form of tracheids (Figure 1A). The appear—
ance of the new xylem (nxy) is shown in Figure lI.

Graft Union Tissue Structure

Concurrent with developmental sequence studies, were detailed
observations of mixed stock and scion tissue in the graft union.
Although this study neared the limits of resolution for the light
microscope, some revealing observations were made.

The common walls of mixed tissue tended to remain un-modified.
Walls of these heterogeneous cells appeared to have normal secondary

wall thickenings and middle lamellae, resulting in a four layered

structure (arrows, Figure 2E and F).

 

24

Heavy pitting occurred in mixed stock and scion cells at the point
of union; a characteristic noted in both self and interclonal grafts
(Figure 3A). At higher magnifications, heavy pitting was observed in
the early formed cambial derivitives (Figure 3B) as well as in later
formed graft union tissue (Figure 3C). These observations were contra—
dictory to those of Preston (1955) who reported pits lacking or diffi—
cult to observe. Such a lack of pitting might only be expected in
parenchymatous cells containing only pit fields; structures less easily
observed.

The significance of heavy pitting at the graft union is a matter
of conjecture, but it seems plausible that it may be an adaptive
mechanism by which the grafted plant may overcome the reported problem
of restricted flow through the graft union (Roach, 1931; Colwell, 1942;
Gur & Samish, 1965). If pits remain constant in number and distribu—
tion during cell extension (Wardrop, 1955), those produced independently
by stock and scion but in association with one another would be expected
to align their pit pairs only at random. Such random alignment would
reduce efficiency of translocation. Therefore, an increase in pit
numbers would increase the probability of pit pair alignment.

Less noticeable were the presence of what appeared to be blind
pits which were believed associated with common walls of stock and
scion (Figure 3D). By reason of random pit alignment, the occurrence
of pits permeating the wall of one graft partner and terminating against

a non-pitted site on the wall of the adjoining cell would be expected.

25

Figure 2 — Fine structure and origin of graft union tissues.
(A) 'Fountain'/'Hetzii', 20 days old, cross-section (X 400).
Proliferation of medullary ray cells of Cambium and at the
graft incision (gi) Cells arising from medullary rays shown
at arrows. (B) 'Fountain'/'Pfitzeriana Kallay', 40 days old,
cross-section (X 413). New vascular derivatives coursing
over injured xylem surface at graft incision (gi).
(C) 'Fountain'/'Fountain', 40 days old, cross-section (X 100).
New vascular tissue over—walling graft incision. Advanced
stage of illustration B. (D) 'Fountain'/'Pfitzeriana Kallay',
50 days old, longitudinal section (X 1000). Detail of common
cell walls of stock and scion. Arrows showing paired, double
secondary wall structures of mixed graft tissue.
(E) 'Fountain'/'Hetzii', 60 days old, longitudinal section
(X 1000). Paired wall structure of adjoining stock and scion
ray and tracheid cells. Void between paired wall structure
indicated by arrows. (F) 'Fountain'/'Pfitzeriana Kallay',
60 days old, cross-section (X 1000). Common walls of mixed
new tracheids. Point of cell contact at arrows.

 

 

27

Figure 3 — Pitting behavior among mixed vascular cells of stock and
scion. (A) 'Fountain'/'Hetzii', 40 days old, longitudinal
section (X 100). (B) 'Fountain'/'Fountain', 50 days old,
longitudinal section (X 400). Pitting in partially differ—
entiated, early formed cells at the graft union.

(C) 'Fountain'/'Fountain', 50 days old, longitudinal section
(X 400). Comparison of pitting in mature xylem (mxy) and
new, recently differentiated xylem (nxy) at the graft union.
(D) 'Fountain'/'Pfitzeriana Kallay', 60 days old, longitudi—
nal section (X 825). Occurrence of "blind pits” in common
walls of new stock and scion tracheids.

28

 

 

 

29

Summary

Anatomical and graft healing sequence studies of three clonal
junipers differing in chromosome number (Juniperus horizontalis
'Fountain', 2n=22; Juniperus chinensis 'Hetzii', 3n=33; and Juniperus
chinensis 'Pfitzeriana Kallay', 4n=44) revealed differing basic
anatomy and established the typical structural changes occurring
during initial stages of graft union development. Also of interest
were observations of pitting behavior at the graft union.

Anatomical study of the three junipers revealed visible differ—
ences in tangential medullary ray cell diameter; this dimension
increasing with rising ploidy level.

Interclonal grafts were studied histologically at 10 day inter—
vals for 60 days from time of grafting to determine possible differ—
ences in their behavior during healing. Although there were no basic
differences in the developmental sequence during this period, the
rates at which these stages appeared was variable among graft combina—
tions.

Between the 10th and 20th day following grafting, voids between
stock and scion began to fill with callus. This was first observed in
grafts involving 'Fountain' and last in those containing 'Pfitzeriana
Kallay' as a component.

Isodiametric cells from uninjured cambia appeared by the 20th
day except in grafts with 'Pfitzeriana Kallay' as the scion. These
Cells by a series of tangential, radial, then tangential divisions

Over—walled the injured surfaces. Interclonal grafts with 'Pfitzeriana'

30
Kallay' as a scion did not produce this tissue until 30 days.

Overwalling of the graft incision was most pronounced in unions
not in close contact, the process resembled surface wound healing.
Overwalling did not occur in closely matched grafts.

Between 40 and 50 days, the graft union, by radial divisions of
the overwalling cells, began filling the area between graft partners
with well organized tissue while crushing existing callus. In
contrast to previously formed tissue, these cells were produced
radially with respect to the graft component from which it arose.

During the 50 to 60 day period a mixing of tissue occurred at
points opposite the cambial rings of stock and scion. These cells
lost their isodiametric shape and assumed the elongated form of
tracheids. Subsequent to this ”cambial bridging”, new xylem formed
in normal centrifugal direction with relation to the total graft union.

Newly formed tissue in a typical graft arose principally from the
stock prior to the 50th day following grafting. After 60 days, con-
tribution of new tissue was nearly equal from stock and scion.

Within mixed graft tissue the walls of adjoining stock and scion
cells were largely un—modified. Typical cell pairs appeared as paired
double wall structures, each with middle lamellas and secondary wall
thickenings. Abnormally large numbers of pits were observed in graft
union tissues; a condition suggested to be an adaptive mechanism by
Which reduced translocation could be corrected through increased
probability of random pit pairing. ”Blind pits" were occasionally
Observed and appeared to be the result of un—aligned pit pairs of adja—

cent stock and scion cells.

 

 

31

Literature Cited

Barker, W. G. 1953. Proliferative capacity of the medullary sheath
region in the stem of Tilia americana. Amer. J. Bot. 40:773-778.

Bradford, F. C. and B. G. Sitton. 1929. Defective graft unions in
the apple and the pear. Mich. Agr. Exp. Sta. Tech. Bul. 99:106 pp.

Braun, H. J. 1961. Recent knowledge of the processes involved in the
grafting of trees. II. Watersupply of scions. Mitt. Deut. Dendrol.
Ges. 62:50—53.

Buck, J. 1954. The histology of the bud graft union in roses. Iowa
State College J. Sci. 28:(4) 587—602.

Camas, G. 1949. Réchérches sur 1e role des bourgeons dans les
phénoménes de morphogenése. Rev. Cytol. Biol. Vegetales. 11:1—195.

Colwell, R. M. 1942. The use of radio—active phosphorus in translo-
cation studies. Amer. J. Bot. 29:789—807.

Evans, G. E. 1969. Developmental biology of interclonal Juniperus
L. grafts. Ph.D. Thesis. Michigan State University.

Gautheret, R. J. 1966. Factors affecting differentiation of plant
tissues grown in vitro. In: Cell differentiation and morphogenesis,
International Lecture Course. Wageningen, The Netherlands. April
26—29, 1965. John Wiley and Sons, N.Y.

Gur, A. and R. M. Samish. 1965. The relationship between growth
curves, carbohydrate distribution and compatibility of pear trees
grafted on Quince rootstocks. Hort. Res. 5:81—100.

Jensen, W. A. 1962. Botanical histochemistry. W. H. Freeman & Co.,
San Francisco, California.

Juliano, J. B. 1941. Callus development in the graft union.
Phillipine J. Sci. 75:245—254.

Kac, V. 1931. Roubovani ovocnych rostlin Vyrocni Zprava Zemskeho
Pomologickeho Ustavu v Praze—Troji. III:152 (as seen in Biol. Abs.
8:118—119, 1932).

Kostoff, D. 1928. Studies on callus tissue. Amer. J. Bot. 15:
565-567.

Kuroda, K. 1960a. Phenomenes d'inductions histogénétiques provoqués
par des formation cribro—vasculaires dans des tissus de carotte
cultives in vitro. Compt. Rend. Acad. Sci. 250:582—584.

32

Kuroda, K. 1960b. Action inductrice des formations cribro—
vasculaires sur les phenomenes d'histogenese dans des tissus de
carotte cultivés in vitro. Compt. Rend. Acad. Sci. 251:272-274.

Mathes, M. C. 1967. The in vitro growth of Acer saccharum and Acer
pennsylvanicum callus tissue. Can. J. Bot. 45:2195-2200.

 

Mergen, F. 1955. Anatomical study of slash pine graft unions.
Quart. J. Florida Acad. Sci. 17 (4):237—245.

Mosse, B. 1962. Graft—incompatibility in fruit trees with particu-
lar reference to underlying causes. Technical Comm. No. 28,
Commonwealth Bureau of Horticulture and Plantation Crops. East
Malling, Maidstone, Kent.

Mosse, B. and M. V. Labern. 1960. The structure and development of
vascular nodules in apple bud-unions. Ann. Bot. 24:500-507.

Muzik, T. J. 1958. Role of parenchyma cells in graft unions in the
Vanilla orchid. Science 127:

Preston, R. D. 1955. Microscopic structure of plant cell walls.
Handbuch der Planzenphysiol. 1:722-730.

Roach, W. A. 1931. The chemistry of the rootstock-scion effect I.
The elements absorbed from the soil. Progress Report. Rep. E.
Malling Res. Sta. for 1928—30. II. Supplement, 101—104.

Sass, J. E. 1932. Formation of callus knots on apple grafts as
related to the histology of the graft union. Bot. Gaz. 94:364-380.

Sharples, A. and H. Gunnery. 1933. Callus formation in Hibiscus
Rosa—sinensis L. and Hevea brasiliensis. Mfill. Arg. Ann. Bot.
47:827-839.

 

Wardrop, A. B. 1955. The mechanism of surface growth in parenchyma
0f Avena coleoptiles. Australian J. Bot. 3:137—147.

Waugh, F. A. 1904. The graft union. Mass. Agr. Exp. Sta. Tech.
Bul. 2:16 pp.

Wilson, K. 1957. Extension growth in primary cell walls with special
reference to Elodea canadensis. Ann. Bot. (n.s.) 21:1—11.

White, P. R. 1964. Some basic parameters in the cultivation of
Spruce tissues. Physiol. Plantarum. 17:600-619.

White, P. R. 1967. Sites of callus production in primary explants
of spruce tissue. Amer. J. Bot. 54:1055—1059.

SECT ION III

 

 

 

33
Distribution of Calcium, Potassium, and Magnesium in
Interclonal Grafts of Juniperus L.

Since before the turn of the century investigations have been
aimed at the mode of mineral translocation in intact plant tissues
(Clements and Engard, 1938; Phillis and Mason, 1940; Stout and
Hoagland, 1939). In grafted tissue, however, this is primarily a
matter of speculation.

First reference made to the relationship of grafting to mineral
transport came when plant physiologists were proposing that water and
minerals moved freely through the plant in a semi—closed system. Hales
(1769) argued that, "The instance of the ilex upon the English oak,
seems to afford a very considerable argument against circulation; for,
if there were a free uniform circulation of the sap through the oak
and ilex, why should the leaves of the oak fall in winter, and not
those of the ilex”.

Near the turn of the century, it had been generally accepted that
circulation in plant tissues occurred through two distinctly different
tissue systems, the xylem and phloem; the former being primarily
responsible for water and mineral transport in at least an acropetal
direction and the latter for basipetal transport of photosynthates and
minerals. Early evidence for water and mineral translocation through
the xylem resulted from ringing experiments with woody perennial plants
Oflalpighi, 1675 and 1679; Clements and Engard, 1938; Phillis and Mason,
1940). In these studies, all tissue exterior to the xylem was removed
armlthe subsequent response observed. No water stress was observable

in plants thus treated until the xylem was also severed.

34

Radioactive isotope work by Stout and Hoagland (1939) illustrated
that element movement occurred primarily in the xylem but that compara-
tively free lateral movement between xylem and phloem also occurred.

Harvey (1931) demonstrated that not only did water move in the
xylem, but that it was limited to the newly differentiated vessel
elements. It was, therefore, concluded that scions could not directly
utilize old xylem elements.

The relationship of water movement through graft unions and ulti—
mate compatibility have, for years, been the object of study. Some
investigators considered restricted water movement across the graft
union to be one of a limited number of prime symptoms of incompatibility
(Chang, 1937; Randahawa & Upshall, 1949; Evans & Hilton, 1955; Evans &
Hilton, 1957; Braun, 1961). Brown (1961) described the scion as
experiencing two periods in its water regime; 1) utilization of water
present in the scion and 2) utilization of water traversing the union
from the stock. Evans and Hilton (1957) found significant differences
in rate of water flow to scions grafted on different apple varieties.

Most translocation studies have been done with radioactive phos—
phorus. In studies using ringed squash plants, Colwell (1942)
demonstrated that basipetally translocated phosphorus accumulated above
the deleted bark ring, in both phloem and xylem, and thereby concluded
that lateral translocation of phosphorus occurred. Dickenson and
Samuels (1956) also demonstrated downward blockage of phosphorus in
apple graft phloem .

Daniel and Thomas (1902) demonstrated that the rootstocks of

intervarietal bean grafts absorbed less than intact plants of either

.._

35

variety. Roach (1931), using Lanes' Prince Albert apple budded on
dwarfing (EM IX) versus non—dwarfing (EM XII) understocks, found that
only molybdenum and lead of the twenty—one elements analyzed were not
transported through the union. Bukovac, Wittwer, and Tukey (1958)
found 45Ca and 32P to be translocated through apple grafts, with no
accumulation at the union. Similarly they found no blockage to occur
in well made tomato self—grafts (1957). These data indicate that most
elements are potentially translocatable through graft unions.

inc was found by Gur and Samish (1965) to accumulate at the
union of 10 year old pear on quince rootstocks. This resulted from
the girdling effect of uncongenial unions.

Present evidence suggests that uptake and translocation of minerals
may be controlled to some extent by the understock used (Warne and
Wallace, 1935). The calcium content in leaves of apple cultivars
grafted on Hibernal was higher than when grafted on French crab root-
stock (Sistrunk & Campbell, 1966). Berry (1939) suggested that a close
correlation may exist between understock vigor and mineral uptake.

Mason and Maskell (1931) found calcium and phosphorus to accumulate
in significant amounts above the bark ring in cotton plants, whereas

INDtassium was not found to accumulate.
The objective of the present study was to:

a. Study calcium, potassium and magnesium distribution at, and
in both components of self grafted plants.
b. Study distribution of these minerals in graft components of

interclonal grafts.

c. Study changes in balance of these elements in self and inter—

clonal grafts as affected by time after grafting.

 

36
Materials and Methods

Experiment I:

Vegetatively propagated clonesl/ of Juniperus horizontalis
'Plumosa Compacta', Juniperus chinensis 'Hetzii', and Juniperus sabina
'Von Ehron', were chosen as biological material for this study. The
plants were approximately one year from cuttings, and 1/8 to 1/4 inch
in stem diameter. Scion wood of equivalent age and from the same plants
as the understocks, was held at 35 C until time of grafting.

Between May 24 and May 28, 1966, the junipers were grafted, using

a modified side—veneer graft (Hartmann and Kester, 1968) in nine combi—

nations:
Scion Stock
1. J. chinensis 'Hetzii' J. chinensis 'Hetzii'
2. J. chinensis 'Hetzii' J. sabina 'Von Ehron'
3. J. chinensis 'Hetzii' J. horizontalis 'Plumosa Compacta'
4. J. sabina 'Von Ehron' J. chinensis 'Hetzii'

5. J. horizontalis 'Plumosa Compacta' J. chinensis 'Hetzii'
6. J. sabina 'Von Ehron' J. sabina 'Von Ehron'
7. J. horizontalis 'Plumosa Compacta' J. sabina 'Von Ehron'

8. J. horizontalis 'Plumosa Compacta' J. horizontalis 'Plumosa Compacta'
9. J. sabina 'Von Ehron' J. horizontalis 'Plumosa Compacta'
All graft unions were tied with 4 inch rubber grafting strips,
Péieked with moist Sphagnum moss, and cultured under a 4 mil polyethylene

Ca-I‘Lopy 24 inches above the plants during the period of investigation.

1/
" IVIOnrovia Nursery Co., Azuza, California

37
One-half of the understock plant was removed 6 weeks following grafting
and the remainder removed 3 weeks later.

Ten collections of 3 samples of each combination were made at 5
day intervals starting June 2, 1966. Stocks and scions were harvested
separately, oven dried, and analyzed for calcium, potassium, and magne—
sium using standard dry ashing and photometry tests (Greweling, 1960).
Results are expressed as stock—scion ratios based on each elements'

percentage by weight of sample tested.

Experiment II:

Plant materials for this phase of the investigation included
Juniperus horizontalis 'Fountain', Juniperus chinensis 'Hetzii', and
Juniperus chinensis 'Pfitzeriana Kallay'. The plants were one year
old, clonally propagatedgé had stem diameters of 3/32 inch to 1/8 inch.
Scion wood in clonal form and the same age as the understocks upon
arrival were held at 35 C until grafting. Using grafting and culture

techniques like those of Experiment I, the following graft combinations

Were made between January 14 and January 16, 1967:

Scion Stock
1- J. chinensis 'Hetzii' J. chinensis 'Hetzii'
2- J. horizontalis 'Fountain' J. chinensis 'Hetzii'

3~ J. chinensis 'Pfitzeriana Kallay' J. chinensis 'Hetzii'
4- J. chinensis 'Pfitzeriana Kallay' J. chinensis 'Pfitzeriana Kallay'
5. J. chinensis 'Hetzii' J. chinensis 'Pfitzeriana Kallay'

6- J. horizontalis 'Fountain' J. chinensis 'Pfitzeriana Kallay'

Z/Pr‘ovided by D. Hill Nursery Co., Dundee, Illinois

38
7. J. horizontalis 'Fountain' J. horizontalis 'Fountain'
8. J. chinensis 'Hetzii' J. horizontalis 'Fountain'
9. J. chinensis 'Pfitzeriana Kallay' J. horizontalis 'Fountain'

At six 10 day intervals, following grafting, three 3/4 inch stem
segments containing the graft union were collected for each combina—
tion, and immediately frozen in polyethylene bags.

Prior to histochemical study, the tissue was thawed and infiltrated
with Tissue-Teci/ embedding medium under vacuum in a venturi type aspira—
tor. The infiltrated material was mounted and frozen in the same media,
sectioned longitudinally and transversely at 18 microns using a refri—
gerated microtome (cryostat). Serial sections were affixed to highly
polished pure aluminum discs (1/8 inch thick by 1 1/4 inch diameter)
for subsequent analysis. The mounting medium served as an adhesive.

The samples were analyzed for calcium, potassium and magnesium
content using an ARLE/ electron microprobe X—ray analyzer, Model
EMX—SM. This instrument combines the advantages of a scanning electron
microscope with X—ray spectrographic analysis. Instrument conditions
Were 14.5 KV accelerating voltage and 0.026 microamperes sample current
resulting in an electron beam diameter of approximately 0.5 microns.
Approximate penetration of the electron beam into the material was 15
microns .

Microprobe analyses of the elements in question were made using

liirie scans across the sample. The X—rays (KG radiations) with charac—

0
teaI'istic wave lengths for each element (Calcium, 3.359 A; potassium,

gt: Ames Company, Elkhart, Indiana
-— .Applied Research Laboratories

L

39
3.742 A; magnesium, 9.889 A) were displayed and photographed from a
cathode ray tube (CRT) or recorded on an X,Y recorder.

Oscillograms (photographs) were prepared from images produced on
the CRT screen by secondary electron detection. The oscillograms,
recorded on Polaroid film, delineated the exact area being examined
as well as the position of the line scan on the sample. In all cases,
magnification was 250 diameters (40 microns per centimeter). Direct
comparisons of the ocsillograms of the tissue with the curves from the
X,Y recorder resulted in precise element localization.

Semi-quantitative information from the line scan data was
obtained by determining the area beneath the curves with the aid of a
planimeter. Comparison of the appropriate oscillogram and its
corresponding sample allowed categorization of each curve with respect
to the tissue through which the analysis was made, thus making it
possible to semi—quantitate the mineral level for each tissue, for
each date, and for each graft combination. The quantity of each
tissue was not constant from sample to sample; therefore, all plani—
meter values were converted to counts per centimeter of sample

traversed by the line scan.

Results and Discussion
..EXE eriment 1:
Results of plant ashing studies, expressed as stock—scion ratios,
aIWE presented in Tables 1, 2 and 3. Statistical analysis of the data
111 Table 1 (Appendix Table B1) for calcium indicated no significant

differences among dates or interactions between dates and graft

 

40

combinations. Combinations were, however, significant at the 1 percent

level.
The mean stock-scion ratio for calcium (Table l) fell between
Although Bukovac gpflgl. (1958) did

those for the other two elements.

not observe blockage of Ca45 in apple grafts, differences in methods

of analysis and plant taxa may explain lack of correlation of their

results with those obtained in this study.
Examination of the means for graft combinations (Table 1) revealed

that calcium accumulated primarily in understocks except when Juniperus

chinensis 'Hetzii' was used as a scion. Self grafts of this clone accu-

mulated significantly greater amounts than the other self grafts which

contained statistically equivalent amounts. The observed consistent

calcium concentration in the graft partner containing this clone lead
to the assumption that either 1) its calcium requirements exceed that
of the other clones or 2) it tends toward "luxury consumption".

Lack of significant differences between values for each clone
self grafted and grafted on other clones suggested that calcium trans-
location was not influenced by the stock used.

Statistical analysis of the potassium data of Table 2 (Appendix

TFable B1) indicated greater stock accumulation than was observed for

magnesium and calcium. Similar to calcium, the ratios were not signi-
ficantly different for dates, or dates times combination interaction,

Whereas, graft combinations were significantly different at the 1

pe rcent level .

Contrary to the calcium data 'Plumosa Compacta' self grafts

accumulated significantly less potassium in the understocks than did

k

 
 

41

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44
'Von Ehron and Hetzii'. Like calcium, potassium translocation was not
significantly affected by understock used. Unlike calcium, all combi—
nations accumulated potassium in the understock.

The mean of magnesium for all combinations (Table 3) was close to
1:00, suggesting that its movement through the union was relatively un—
impeded. Four of the nine combinations accumulated magnesium in the
scion; all others contained higher amounts in the rootstock. All self
grafts distributed magnesium in similar fashion, having stock—scion
ratios close to 1:00. Combinations involving 'Hetzii' as a scion had
higher amounts of magnesium in rootstocks than other heterografts
which tended to accumulate magnesium in the scion. 'Von Ehron' as a
scion on 'Hetzii' rootstocks contained the highest concentration of
magnesium when compared to other combinations. This constituted the
lowest observed stock—scion ratio for all combinations and elements
(0.52).

Examination of significant magnesium stock-scion means across
dates revealed that 12 day grafts had more magnesium in the scion than
at any other date. This was the only date that all graft combinations
contained higher amounts of magnesium in scions than in stocks. This
observation could be either a measure of rootstock depletion or the
first date at which magnesium traversed the graft union.

A significant interaction between graft combinations and dates
indicated that magnesium distribution varied among graft combinations
at a given date. Greatest deviations occured in 'Hetzii' on 'Plumosa
Compacta' grafts during the 1st, 3rd, 4th and 6th harvest dates when

there was a high accumulation in the understock.

45

In certain interclonal combinations and their reciprocals magne—
sium concentration appeared to be associated with only one partner of
the graft. This is evident with grafts of 'Hetzii' on 'Von Ehron' and,
with exception of two dates, 'Plumosa Compacta' on 'Hetzii' (Table 3).
'Hetzii', being common to these combinations, may suggest a magnesium
balance which is influenced by the nutritional status of this clone.

Comparison of grand means revealed that potassium and calcium
accumulated in the understock, while magnesium appeared evenly distri—
buted. The relatively slow mobility of calcium was also demonstrated
by Biddulph E£.§1' (1958) in studies of translocation in bean grafts
where calcium was shown to be less mobile than sulphur or phosphorus.
Experiment 2:

Data obtained from electron microprobe X—ray analysis for calcium,
potassium and magnesium distribution was accumulated graphically and
converted to numerical values based upon the area underneath the curves.
Figure 1 illustrates a typical curve and oscillogram delineating the
tissue through which the analysis was run. These data, although
incomplete, provide certain significant information. Whereas, analyti—
cal techniques for Experiment 1 allowed for comparisons of mineral
distribution between graft components, microprobe analysis made possible
detection and distribution of minerals within individual tissues of the
graft union.

If mineral content of the xylem is taken to represent transitory
materials, stock accumulation (Figure 2) indicates that calcium passes
least readily through the graft union. Potassium and magnesium

appeared to move with relative ease as indicated by generally equal

46

Figure 1. Line profile analysis of a juniper graft union showing
A) distribution of K. Mg and Ca; and B) secondary electron
oscillogram with line scan from 1 to 2. Magnification
X500.

UM

47

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I

 

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me

XYLEM

PHLOEM

CORTEX

2. Mean calcium, potassium and magnesium content of inter—
clonal juniper graft tissues
asured by electron microprobe X-ray analysis.

within stock and scion as

49
stock-scion balance in the xylem.

With the exception of magnesium, mineral distribution in the
phloem was balanced between stock and scion and may represent base
levels for this tissue. Magnesium content of the stock phloem was
greater than that observed in xylem of the stock; the possible result
of accumulation and lateral transport from the xylem.

In cortical tissue, calcium was present in greater amounts in the
scion than in the stock, while potassium and magnesium were distri-
buted equally between graft components. The excess amount of
calcium present in the scion may have been 1) readily transported
laterally in the stock to the cortex where it moved acropetally into
the scion, or 2) readily translocated across the union in the xylem,
then transported laterally to the cortex. The unrestricted transloca-
tion of this element observed by Bukovac g£_§l, (1958) in apple grafts
suggests the second hypothesis to be the more valid.

Unlike calcium, scion potassium levels were greater in xylem than
in phloem or cortical tissue while magnesium levels were equivalent in
xylem and phloem but much higher in the cortex. The observed cortical
tissue levels of calcium and magnesium may represent differences in
structural element levels for these tissues or storage resulting from
"luxury consumption”.

Summary

Calcium, potassium and magnesium content of interclonal juniper

grafts were determined in two separate experiments, using ashing—

photometry tests and electron microprobe X-ray analysis.

50
Plant Ashing and Photometry

Accumulation of solutes in understocks, expressed as stock—scion
ratios obtained from separate ashing of entire stocks and scions,
occurred in decreasing order; potassium, calcium, and magnesium.

'Hetzii', as a scion, accumulated more calcium than scions from
other clones. While no difference was found for 'Von Ehron' or
'Hetzii' selfgrafts, those of 'Plumosa Compacta' accumulated small
amounts of potassium in the understock. Grafts involving 'Hetzii'
accumulated more calcium than potassium in the scion.

Mean stock-scion ratios of 1.00 for magnesium, suggested that
this element moved relatively unrestricted across the graft union in
comparison to calcium or potassium. More magnesium was found in
understocks of grafts with Juniperus chinensis 'Hetzii' scions than
in all other combinations. Juniperus sabina 'Von Ehron' as the scion,
accumulated more magnesium than any other combination. A11 grafts
after 12 days contained more magnesium in the scion than at any other
time.

In graft combinations with Juniperus chinensis 'Hetzii' low
magnesium levels were evident whether it was used as stock or scion.
This suggests that a lower magnesium level may be in this clone.

The fact that more calcium and potassium accumulated in under—
stocks than magnesium suggested that differences existed in translo—
cation rates across the graft union.

Microprobe Analysis:
Considering xylem concentration as a measure of mobile elements,

calcium appeared to accumulate in the understock while potassium and

51
magnesium moved readily through the graft union.

In phloem tissue, distribution was generally equal between stock
and scion for all elements; magnesium being slightly greater in the
stock.

Though potassium and magnesium were found equally distributed
between stock and scion in the cortex, calcium accumulated in greater
amounts in the scion. Because of its apparent blockage at the xylem
union, excess scion calcium in the cortex was thought to result from
lateral stock transport to the cortex where it was able to diffuse
through the union; and accumulate in possible "luxury" amounts in
the scion.

Though only general comparisons were possible, it was interesting
to note that results obtained from ashing — photometry and from elec—
tron microprobe were quite different. Ashing and photometry data, a
measure of entire stock or scion mineral composition, showed under—
stock accumulation to be, in decreasing order, potassium, calcium
and magnesium. Microprobe data, concerning the graft union only,
indicated the apparent decreasing order of understock accumulation to
be calcium, potassium, and magnesium. Thus, ashing data may represent
element composition of the intact graft partners, while electron micro—
probe values reveal apparent rates of element movement through the

graft union.

52

Literature Cited

Berry, W. E. 1938. A note on the relation of absorption to vigour
in apple stocks. Ann. Rep. Long Ashton Res. St. for 1938, 63—65.

Biddulph, 0., S. Biddulph, R. Cory, and H. Koontz. 1958. Circulation
patterns for phosphorus, sulphur, and calcium in the bean plant.
Plant Physiol. 33: 293—300.

Braun, H. J. 1961. Recent knowledge of the processes involved in
the grafting of trees. 11. Water supply of scions. Mitt. Deut.
Dendrol. Ges. 62:50—53.

Bukovac, M. J., H. B. Tukey, and S. H. Wittwer. 1957. Effet de la
greffe et 1'orientation du scion sur la circulation des substances
nutritives d'apres la méthode des radio—isotopes. In: Colloque
International sur la Greffe de Tissus Végetaux et Animaux. Comptes
Rendus des Travaux Rennes, France Juin 7-11, 1957. pp. 115—130.
(In French).

Bukovac, M. J., S. W. Wittwer and H. B. Tukey. 1958. Effect of stock—
scion interrelationships on the transport of 32P and ”SCa in the
apple. J. Hort. Sci. 33:145—152.

Chang, Wen-Tsai. 1937. Studies in incompatibility between stock and
scion with special reference to certain deciduous fruit trees.
Jour. Pom. and Hort. Sci. 15:267—325.

Clements, H. F. and C. J. Engard. 1938. Upward movement of inorganic
solutes as affected by a girdle. Plant Physiol. 13:103—122.

Colwell, R. M. 1942. The use of radioactive phosphorus in translo—
cation studies. Amer. Jour. Bot. 29:789—807.

Daniel, L. and V. Thomas. 1902. Sur l'utilisation des principes
mineraux par les plantes greffees. Bull. Soc. Scient. et medicale
de 1'ouest. 11:405—409.

Evans, W. D. and R. J. Hilton. 1955. Methods of evaluating stock/
scion compatibility in apple trees. Abst. in Agric., Inst. Rev.
10 (3):35.

Evans, W. D. and R. J. Hilton. 1957. Methods of evaluation of stock/
scion compatibility in apple trees. Canad. J. Plant Sci. 37:327—336.

Greweling, T. 1960. Mineographed procedures for chemical analysis of
plant materials, Agronomy Department, Cornell University, 40 pp.

53

Gur, A. and R. M. Samish. 1965. The relationship between growth
curves, carbohydrate distribution and compatibility of pear trees
grafted on quince rootstocks. Hort. Res. 5:81-100.

Hales, S. 1769. Vegetable staticks. 1726-7 Vol. I. Edition 3
London; As seen in: Crafts, A. 1961. Translocation in plants.
Holt, Rinehart and Winston, Inc., New York.

Hartman, H. T. and D. E. Kester. 1968. Plant Propagation, Principles
and Practices. Second Edition, Prentice-Hall, Englewood Cliffs,
N. J.

Harvey, E. M. 1931. A method for studying water conduction in plants
in relation to pruning, grafting, and other horticultural practices.
Ore. Agr. Exp. Sta. Bul. 279.

Malpighi, Marcellus. 1675 and 1679. Die Anatomie der Pflanzen,
Leipzig:Wilhelm Engelman. As seen in: Crafts, A. S. 1967. Trans—
location in plants. Holt, Rinehart and Winston, Inc., New York.

Mason, T. G. and E. J. Maskell. 1931. Further studies on transport
in the cotton plant 1. Preliminary observations on the transport
of phosphorus, potassium and calcium. Ann. Bot. 45:125-173.

Phillis, E. and T. G. Mason. 1940. The effect of ringing on the
upward movement of solutes from the roots. Ann. Bot. 4:635—644.

Randahawa, G. S. and W. H. Upshall. 1949. Congeniality of some pear
varieties on quince A. Sci. Agric. 29:490-493.

Roach, W. A. 1931. The chemistry of the rootstock-scion effect. I.
The elements absorbed from the soil. A progress report. Rep. East
Malling Res. Sta. for 1928-1930. 11. Supplement, 101-104.

Sistrunk, J. W. and R. W. Campbell. 1966. Calcium content differences
in various apple cultivars as affected by rootstock. Proc. Amer.
Soc. Hort. Sci. 88:38—40.

Stout, P. R. and D. R. Hoagland. 1939. Upward and lateral movement of
salt in certain plants as indicated in radioactive isotOpes of
potassium, sodium and phosphorus absorbed by roots. Amer. J. Bot.
26:320-324.

Warne, L. G. G. and T. Wallace. 1935. The composition of the terminal
shoots and fruits of two varieties of apple in relation to rootstock
effects. J. Pomol. 13:1—31.

SECTION IV

54
Serological Determination of Tissue Contribution from Stock and Scion in
Juniperus L. Grafts
Introduction

Studies of mixed graft union tissue have been limited by an in—
ability to identify cells derived from each of the graft components.
Immunological technqies offer promising new ways to overcome this
problem. This approach has been used by plant taxonomists to ascertain
botanical relationships using protein similarity as a basis for separa—
tion of taxa. It has been possible, therefore, to suggest systematic
changes in such woody perennial genera and families as Quercus and
Castanea (l3), Magnoliaceae (15), Cornaceae and Nyssaceae (8), and in
Solanum (10).

Rives (21,22) used serological techniques to predict the affinity
of one graft partner for another in studies of grape. In this case,
immune precipitin reactions occurred in those varieties characteris—
tically difficult to graft while positive reactions occurred in those
varieties which successfully intergraft.

In similar studies, Green (12) studied serological relationships
among species of six families and two sub-families known to success—
fully intergraft. He demonstrated positive precipitin reactions for
typically successful interspecies grafts, and consistently negative
reactions for non—successful grafts.

Kostoff (17) reported precipitin reactions in graft unions and
concluded that plants were capable of acquiring immunity to foreign
plant protein similar to that occurring in immunized animals. Chester

(3,4) disputed this theory after immune response studies of extracts

55
from more than 30 species and varieties of woody plants failed to
result in precipitin reactions. He suggested that the precipitation
observed by Kostoff was calcium oxalate crystals.

Though antigen extracts used for plant studies have been pre—
pared principally from dried seed (12,13,15), Rives (21,22) and
Tucker (25) obtained favorable responses from shoot tip extracts.

Due to variation from animal to animal in response to injections,
certain workers (1,9) are now using inbred strains of mice instead of
rabbits. Using the Balb/C Jax strain of white mice, Fink and Quinn
(9), found an intraperitoneal injection to yield the greatest amount
of antisera; this has been confirmed by Anacker and Munoz (1).

In recent years a modified antibody technique using fluorescent
labelling has played an important role in histological studies of
animal tissue (5,26,11).

Very little information is available on fluorescent antibody
techniques in plant histochemistry. Coons (5) described its use
for microslide preparations of animal tissues. His procedure included
cryostat sections of fresh frozen tissue; the slide preparations were
incubated with labelled antibody and examined under the ultraviolet
microscope.

The present study was designed to investigate the serological
technique as a tool for studying developing graft unions. Procedures
modified from those used for both plant taxonomy and animal tissue

studies were used as the basis for the experimental technique.

56
Material and Methods
Graft Microslide Prgparation
Three clonal juniper varieties, Juniperus pprizontalis 'Fountain',
Juniperus chinensis 'Hetzii', and Juniperus chinensis 'Pfitzeriana
Kallay', were obtained in October 1966 from a commercial nursery
sourc 1/.
Upon arrival, the understock plants, measuring 1/8 and 3/16
inch diameter, were subjected to cold treatment for approximately 60
days to satisfy their cold requirements. Scion wood, obtained from
the same nursery source, was placed in storage at 350 F. to assure
continued dormancy. On December 10—11 understocks were potted and
benched in a 680 to 700 F. night temperature greenhouse. Between
January 15, and January 17, 1967 a number of the following graft
combinations were prepared using a modified side graft.
Stock Scion
. horizontalis 'Fountain'
chinensis 'Hetzii'
chinensis 'Pfitzeriana Kallay' chinensis 'Pfitzeriana Kallay'

chinensis 'Pfitzeriana Kallay' chinensis 'Hetzii'

. horizontalis 'Fountain' J
J

J

J

chinensis 'Hetzii' J. chinensis 'Pfitzeriana Kallay'
J

J.

J.

J.

chinensis 'Hetzii'

horizontalis 'Fountain' chinensis 'Hetzii'

chinensis 'Hetzii' horizontalis 'Fountain'

. horizontalis 'Fountain' chinensis 'Pfitzeriana Kallay'
chinensis 'Pfitzeriana Kallay' horizontalis 'Fountain'

OGDNO‘Lnwal-l
C—stf—tQC-«LtLtLat—t

All graft unions were covered with damp Sphagnum moss, and the
entire bench covered with 4 mil clear polyethylene supported 24 inches
above the plants on a wooden frame.

Using standard propagation practices, the understock plants were

cut back to one—half their mass 40 days after grafting; the remainder

l/D. Hill Nursery Co., Dundee, Ill.

57
removed at the union 60 days after grafting.

For serological studies, stem segments encompassing the graft
union were collected at random. Samples of each combination were
collected from 10 to 60 days after grafting. The graft unions were
placed in refrigerated storage (—220C) in polyethylene bags to await
sectioning.

The samples were evacuated and infiltrated for one hour in a
1.5% solution of Reteng/ using a venturi type vacuum aspirator.
Sections were cut at 10 microns using a Model CTD refrigerated
microtome (crystat)§/ at a cutting temperature of —150 C. Sections
were picked up directly from the microtome blade using a thin film
of 75% Reten on a microslide previously coated with ARG adhesive
Jensen (14).

Antibody Preparation

Animal variation was minimized by using an inbred strain of
mice, Balb/C Jax, for immunization. Three month old female mice
were injected with antigen from each of the two junipers studied.
Although sex had no effect, Fink and Quinn (9) reported that four
to five month old animals produced more antibody than younger ones.
The animals were reared in an air conditioned animal room under
carefully controlled, sterile conditionsfl/.

Antigen sera was prepared from leaf and succulent stem tissue.
This material, upon removal from the plant, was freeze—dried in a
2/A catonic, water soluble polymer, product of Hercules Powder Co.
é/International Equipment Company.
4/Animals, material and related facilities provided for under

National Science Foundation Grant Number G.B. 2714 supporting
serological studies of Agropyron spp.

 

58
refrigerated lyOphilizer and ground in a ballmill. Since differing
species might elicit differing antibody responses, the tetraploid

Juniperus chinensis 'Pfitzeriana Kallay' and the diploid Juniperus

 

horizontalis 'Fountain' were chosen as antigen sources.

 

Protein was extracted from plant tissue powder by suspending
500 mg tissue in 4 ml of 70% ethanol for 4.5 hours at 680 C. After
a 5 minute centrifugation at 3,000 r.p.m. in a clinical centrifuge,
the supernatant was held at 40 C. overnight, then recentrifuged at
2600 g. The resulting supernatant was freeze-dried.

A micro-Kjeldahl test was used to determine nitrogen concentra-
tions of protein extracts. Using a factor of 6.0 to convert total
nitrogen values to total protein extracted, a yield of 0.84 mg
protein/ml of solvent was established for Juniperus horizontalis

'Fountain' and 1.83 mg protein/ml solvent from Juniperus chinensis

 

'Pfitzeriana Kallay'. In preparation for immunization, lyophilized
protein was adjusted with physiological saline to a concentration
of 0.50 mg/0.25 cc injection. The antigen preparation was filtered
through a Millipore filter of 0.65 micron pore size to remove con-
taminating bacteria.

Prior to injection the antigen was diluted with an equal
volume of Freund's adjuvant to induce blockage of the lymphatic
system; a procedure necessary to promote antibody accumulation con-
taining ascites fluid in the body cavity. All injections were intra-
peritoneal using a 1 cc syringe and a 1 inch, 18 gauge disposable

needle.

The animals were injected five times at weekly intervals

59

followed by two booster shots. Three animals were immunized for
each antigen used. Failure to accumulate sufficient volume of peri-
toneal fluid after 12 weeks made it necessary to administer an addi-
tional 1/2 cc of Freund's adjuvant.

A microprecipitin titer test was employed to determine antibody
levels of the antisera (2). This procedure involved the reaction 0f
several antisera concentrations with the antigen used to immunize the
animals. Dilutions of 4:1, 8:1, 16:1, 32:1, and 64:1 were prepared
in a porcelain spot plate using a one m1 microsyringe. The tests
included antigen controls, antisera controls, and cross reactions
with antigen from the other clone to measure antibody specificity.
The resulting precipitin reaction was observed under a dissecting
microscope, and the dishes photographed.

Ascites fluid was removed from test animals at several dates
until the minimum necessary volume was obtained. The resulting fluid
was frozen immediately upon extraction.

Antisera were prepared for conjugation using a slight modifica—
tion of the procedure described by Goldman (11), as follows:

1. Removal of albumin
a. To ascites fluid add an equal volume of 0.15 M NaCl.

b. With mechanical stirring in a 40 F. ice bath add slowly an
equal volume of cold saturated (NH4)2504-

c. Continue to stir for 30 minutes.
d. Centrifuge in the cold (4°C.) and retain pellet. A table
model Sorvall Clinical centrifuge at approximately 3500

r.p.m. was used in the present work.

e. Wash, pellet once using cold 1/2 saturated (NH4)2804.

60

f. Re—centrifuge and retain precipitate.

g. Dissolve precipitate in phosphate buffered saline (pH 7.2).
Total volume approximately 1/3 that of the original ascetes
fluid.

Removal of ammonium ions—(NH4)+.

a. Dialyze against 1 liter phosphate buffered saline (PBS) in
the cold (4°C.) with mechanical stirring.

b. Change PBS at 1 hour and 5 hours, then continue dialysis over
night.

Conjugation of antibody protein with fluorescein isothiocyanate

employed the following simplified technique described by Jutila (16).

l.

10.

Determine protein content of immune globulin. In the present study
the micro—Kjeldahl technique was used.

To an Erlenmeyer flask add with mechanical stirring:

a. Physiological saline (0.85%) 10 ml.
b. HCO3-CO3 buffer (0.5 M, pH 9.0) 3 ml.
c. Acetone 2 ml.

Cool in acetone—dry ice bath until crystals form.
Add with stirring, 10 m1. diluted globulin (from 2b above).
Cool with stirring as in 3 above.

Add slowly 1.5 m1 acetone—fluorescein isothiocyanate (0.05 mg/mg
globulin protein).

Stir in the cold (4°C.) for 18 hours.

Dialyze against phosphate buffered saline (0.01 N, pH 7.2) until
saline no longer contains dye.

Filter through 1.5 micron Millipore Filter.

Store for short periods at 4°C. or shell freeze and store at
—70°C. for extended periods.

Prior to reaction of labelled antibody with microslide prepara—

tions, the conjugates were absorbed for 12 hours with Fluorescent

 

61
Antibody (FA) liver powderi/ and centrifuged for 30 minutes in a
refrigerated centrifugeé/ at 22,500 X g; liver extract pellet being
discarded.

Optimum conjugate concentration for microslide labelling was
determined by testing with un—diluted conjugate and conjugate at
1:1 and 2:1 dilution (phosphate buffered saline: conjugate).

A limited quantity of labelled antibody made it possible to
study only 4 of the 9 graft combinations prepared. Because 'Fountain'
on 'Hetzii', 'Hetzii' on 'Fountain', 'Pfitzeriana Kallay' on 'Fountain'
and 'Fountain' on 'Pfitzeriana Kallay' were highly successful grafts
(7), they were chosen for study. In studying the latter two combina-
tions, the preparations were allowed to absorb unlabelled antibody
of one clone, then were incubated with conjugate of the other clone.
This was done to eliminate as much non—specific labelling as possible,
and was an adaptation of a procedure described by Redys g£_a1. (20)
for blocking cross reactions in Group A and Group B streptococci.

Labelling proceeded for 30 minutes in moist blotter lined Petri
dishes, at 37° C. Excess antibody was carefully removed by syringing
with phosphate buffered saline. Coverslips were mounted using a
commercial fluorescent antibody mounting medial/ and the preparations
microscopically examined using a ultra violet light source. Photo—

micrographs were made of significant observations.

5/Difco Laboratories, Detroit, Michigan
Q/International Equipment Co.
l/Difco Laboratories, Detroit, Michigan.

62

Results and Discussion

The results of this investigation indicated that the use of
fluorescent antibody methods appeared to offer a promising histochemi-
cal tool for studies of plant graft union development.

An interesting observation, although only indirectly related to
this study, was made relative to protein concentrations of the juni-
pers used as antigen sources. Several micro—Kjeldahl determinations,
used to arrive at the most efficient extraction technique for antigen,
indicated that 'Fountain' contained a mean of 0.84 mg protein/ml of
extractant as compared to 1.83 mg protein/mg extractant for
'Pfitzeriana Kallay'.

Prior to harvest of the antisera from animals, the sera was
checked for titer level using a microprecipitin test. To check for
suspected non-specificity of antibody production, cross reaction,
using unrelated antigen, was included in the titer test. The
resulting precipitin reactions are illustrated in Figures 1A for
'Fountain' and in 1B for 'Pfitzeriana Kallay'.

Represented in figures 1A and 1B are antigen controls (AC) and
antisera controls (SC) in lower squares, antigen reacted with serially
diluted (4:1, 8:1, 16:1, 32:1, 64:1 physiological saline: antisera)
antisera in the middle squares, and serially diluted antisera reacted
with antigen of the other juniper in the upper squares. The degree
of "milkiness" of spots represents the amount of precipitation brought
about by the antigen—antisera reaction.

Antigen and antisera controls appeared to be un—reacted except

63
for the former in 'Fountain'; this may have resulted from cross con-
tamination. Precipitin reaction for 'Fountain' was more intense than
'Kallay' as indicated by the "milky" appearance. This was believed
due to a higher titer level for 'Fountain'. The strong reactions
were observed at a dilution of 1:64 in 'Fountain' while at 1:32
dilution 'Kallay' faded providing evidence of this difference in
titer levels.

0f greatest significance was the observations that reactions
occurred when antisera was combined with antigen of the juniper not
used to obtain each antisera (top rows of squares, Figure 1A and 1B).
Occurrence of cross reactions strongly suggested immune sera was not
totally clonal specific. Greater specificity, however, was noted in
'Fountain' sera than in that of 'Pfitzeriana Kallay' when intensity
of cross reactions were compared.

Background fluorescence or illumination was measured photographs
ically with ultraviolet light, non—fluorescein labelled xylem and
cortical tissues of clones used as antigen sources (Figure 2). A
comparison of these photomicrographs revealed that even though back-
ground fluorescence was relatively high in both varieties it appeared
to be greater in 'Fountain' than in 'Pfitzeriana Kallay'. The
observed background fluorescence provided confidence that observed
differences between fluorescein antibody labelled and non-specifically
labelled tissues were real.

Figure 3 represents photomicrographic comparisons of fluorescence
intensity between fluorescein labelled and non—labelled xylem, phloem,

and cortical tissue. To obtain accurate comparative measures of

. *1.
h -5. M

~ -_ _ .i
7"“) ~<4o=_ _P «gr

A ’ ,14 am. 32‘ (.7.

 

Figure 1 — Microprecipitin test plates for antisera of Juniperus
horizontalis 'Fountain' (A) and Juniperus chinensis
'Pfitzeriana Kallay' (B). Lower squares: antigen
controls (AC) and antisera controls (SC). Middle
squares: diluted antigen—antibody reactions. Top
squares: diluted cross-reactions, in A with
'Pfitzeriana Kallay' antigen, and in B with
'Fountain' antigen.

65

Figure 2 - Background fluorescence of non-fluorescein labelled xylem
and cortex. (a) Juniperus horizontalis 'Fountain' xylem
and (b) Cortex, (c) Juniperus chinensis 'Pfitzeriana Kallay'
xylem (d) and cortex.

 

67
intensity, U.V. illumination level was left stable with only fields
of observation varying.

Photomicrographs 3A and 3B represent flourescence of scion xylem
and understock xylem respectively, of a 'Fountain' on 'Kallay' graft
having scions labelled with antibody specific for the scion. Figures
3C and 3D represent serial sections of the same graft combinations,
but reacted with 'Pfitzeriana Kallay' antibody. Examination of these
photomicrographs revealed fairly distinct difference in labelling of
xylem between the scion (Figure 3A) and stock (Figure 3B). When stock
labelled, differences were again detected, although in reversed order;
the stock fluorescence (Figure 3D) exceeded that of the scion (Figure
3C). Notable was the observation that degree of contrast between
labelled and unlabelled xylem was much greater when using 'Fountain'
antibody than with that of 'Pfitzeriana'. This, however, was due, in
part, to the greater background fluorescence observed in unlabelled
tissue of 'Fountain'. This was added evidence of greater specificity
of reaction for 'Fountain' than for 'Pfitzeriana'; a condition first
detected in microprecipitin test results (Figure 1).

Although intensity, both total and comparative, was lower in
cortical tissue, differences were apparent between scion labelled
'Fountain' cells (Figure 3D) and understock cells (Figure 3F).

Figures 3G and 3H represent the same graft combinations labelled
with 'Pfitzeriana' antibody. As noted for xylem, the expected
reversal of fluorescence was observed. 'Fountain' antibody appeared
to have less Specificity in this tissue than in xylem, while that of

'Pfitzeriana' remained equivalent for both tissues.

68

Figure 3 - Photomicrographs of fluorescent antibody labelled stocks
and scions of Juniperus horizontalis 'Fountain' on
Juniperus chinensis 'Pfitzeriana Kallay' grafts. A. Xylem
of 'Fountain' scion fluorescein labelled with 'Fountain'
antibody. B. 'Pfitzeriana Kallay' understock of the same
graft. C. Scion xylem of graft labelled with 'Pfitzeriana
Kallay' antibody. D. Understock xylem of the same graft.
E. Cortical tissue of 'Fountain' scion with fluorescein
labelled 'Fountain' antibody. F. Understock cortex of the
same graft. G. Scion cortex of graft labelled with
'Pfitzeriana Kallay' antibody. H. Understock cortex of the
same graft.

 

 

70

Typical examples of fluorescence patterns observed in mixed
tissue of juniper graft unions are shown in Figure 4. These illustra-
tions point to the potential usefulness of fluorescent antibody techni-
ques for identifying tissue belonging to one component of heterogeneous
graft unions.

Figure 4A shows fluorescence labelling of mature stock and scion
xylem tissue; the labelled 'Fountain' scion fluoresces more strongly

than the Juniperus chinensis 'Hetzii' understock. The lack of

 

fluorescence of the intervening cells suggested that they arose from
the understock.

In the cortical tissue of a 'Fountain' on 'Hetzii' graft a more
exacting separation of stock and scion tissue was noted. This is
illustrated in Figure 4B. The broken line represents the approximate
position of the original graft incision.

Figure 4C represents the stock—scion junction of a 'Pfitzeriana'
on 'Fountain' graft stained with standard biological stains and viewed
with-a standard light source. The same graft combination when scion
labelled and viewed under ultraviolet light is illustrated in Figure
4D, where, although the stock portion of the graft is out of plane,
greater fluorescence was observed in the scion. Although much of the
fluorescence appeared to be background or non—antibody, fairly dis—
tinct fluorescent spots were observed in the labelled scion phloem
(upper right corner). Most of the tissue between the stock and scion
appeared to arise from the understock.

Figure 4E orients the fluorescing tissue of the 'Pfitzeriana'

on 'Fountainf graft shown in Figure 4F. Major labelling appeared in

71

Figure 4 — Fluorescent antibody labelling of graft unions. A. Xylem
junction of a scion (sc) labelled Juniperus horizontalis
'Fountain' on Juniperus chinensis 'Hetzii' graft. B. Mixed
stock and scion (sc) cortical tissue of graft described in
A. Graft line indicated by the broken line. C. 'Pfitzeriana
Kallay' on 'Fountain' graft showing point of cambial align-
ment of stock and scion (sc). Graft stained with safranin
and Azur B. D. Graft described in C labelled with fluores-
cent 'Pfitzeriana Kallay' antibody. E. 'Pfitzeriana Kallay'
on 'Fountain' stained with safranin and Azur B. Newly
formed scion xylem nxy (sc) to the right side of the
photomicrograph. F. Serial section of the same graft
labelled with fluorescent antibody of scion. G. Mixed
cortical and epidermal tissue of 'Pfitzeriana Kallay' on
'Fountain' graft stained with safranin and Azur B. Junction
between stock and scion (sc) indicated by broken line.

H. Same section as in G with fluorescein labelled
'Pfitzeriana'Kallay' antibody.

 

 

73

the new scion xylem (right) and what appeared to be an island of
crushed scion callus tissue near the middle of the photomicrograph;
all other tissue having apparently arisen from the stock.

Figures 4G and 4H illustrate labelling patterns in cortical and
cork tissues of a 'Pfitzeriana' on 'Fountain' graft; the dotted line
delineating the original graft incision. Greater fluorescence was
noted in the labelled scion tissue.

Though results using fluorescein labelled antibody were
encouraging, a degree of refinement was deemed necessary to procure
antibody of sufficient specificity to assure accurate separation of
all tissues. The level of background fluorescence seen when un—
labelled tissue was viewed under ultraviolet light (Figure 2)
suggested that labelling was relatively low in certain tissues;
especially xylem.

The fact that protein levels appeared to vary in direct propor—
tion to ploidy level suggests that fluorescence due to protein
specific antibody may be quite low and that any differences between
graft tissue may have resulted from differing amounts of protein
available for labelling rather than due to antisera specificity. Due
to this possibility, techniques should be modified to utilize finite
plant cell fractions as antigens rather than crude plant extract, as
used in the present study.

Summary

Immunization of inbred white mice using Juniperus horizontalis

 

'Fountain' and Juniperus chinensis 'Pfitzeriana Kallay' as antigen

 

sources produced species specific antibody which, when labelled with

74

fluorescein isothiocyanate aided in plant graft studies. Labelled
microslide preparations of interclonal grafts examined under dark—
field illumination and ultraviolet light source, provided good
isolation of tissues derived from each graft component. Major
objective of the study was to determine feasibility of this techni—
que for ascertaining the contribution of the stock and scion to
healing of the graft union.

Microprecipitin reactions of antigen with immune serum resulting
from its introduction into animals revealed highest titer levels in
'Fountain' antisera; good reactions occurred at dilutions of 64:1,
the highest tested. Cross titering of each antisera with antigen of
the other clone indicated fairly high levels of cross reaction, taken
as a measurement of non-specificity of the antibody produced. Because
less intense cross titer reactions were observed using 'Fountain'
antisera, greater antibody specificity was suspected for protein of
this juniper; a supposition born out by the observed reaction when
this antisera was applied to grafts involving 'Fountain' with other
clones.

Examination, under ultraviolet light, of non—antibody treated
microsections indicated the level of background fluorescence to be
relatively high. Greater amounts were observed in 'Fountain' tissues.
Although this observation suggested that clonal differences in fluores-
cence due to antibody labelling were quite small, known background
fluorescence levels provided confidence that any observed differences
in fluorescence between tissues of heterogeneous grafts were real and

due to antibody labelling.

75

When grafts of 'Fountain' on 'Pfitzeriana' were incubated with
'Fountain' antibody, scion xylem and cortical cell fluorescence
exceeded that for the understock with the most obvious differences
occurring in the xylem. When this graft combination was treated with
labelled antibody of the understock, tissues of this graft component
exhibited more fluorescence. Unlike 'Fountain' antibody, specificity
was equally apparent in xylem and cortical cells.

Interclonal graft unions studied by fluorescent antibody
labelling of one graft component was generally found to result in
clear separation of tissues belonging to labelled and unlabelled
components of the graft. Specificity, however, was greater in the
more parenchymatous tissue of the phloem, cortex and epidermis than
in the xylem.

Though results were encouraging, it is felt that use of refined
antigen rather than crude extract would result in antibody production

of greater specificity.

10.

11.

12.

76

Literature Cited

Anacker, R. L. and Munoz, J. Mouse antibody. I. Characteriza-

tion of antibody in mouse peritoneal fluid. J. Immun. 87:
426-432. 1961.

Ball, E. M. Serological tests for the identification of plant
viruses. 16 pp. Publ. by the American Phytopathological
Society. 1961.

Chester, K. S. Graft blight: a disease of lilac related to the
employment of certain understocks in propagation. J. Arn. Arb.
12:78-145. 1931.

Chester, K. S. Studies on the precipitin reaction in plants.
I. The specificity of the normal precipitin reaction.
J. Arn. Arb. 12:52-74.’ 1932.

Coons, A. H. Histochemistry with labelled antibody. Int. Rev.
Cytol. 5:1—23. 1956.

Coons, A. H. Fluorescent antibody methods. In: General
Cytochemical Methods, (J. F. Danielli, ed.) 1:399-422.
Academic Press, N. Y. 1958.

Evans, G. E. Developmental biology of interclonal Juniperus L.
grafts. Ph.D. Thesis, Michigan State University. 1969.

Fairbrothers, D. A. and Johnson, M. A. Comparative serological
studies within families Cornaceae (dogwood) and Nyssaceae
(sourgum). pp. 305-318. In: A.A. Leone (ed) Taxonomic
Biochemistry and Serology. Ronald Press. 1964.

Fink, M. A. and Quinn, V. A. Antibody production in inbred
strains of mice. J. Immun. 70:61-67. 1953.

Cell, P. G. H., Hawkes, J. G. and Wright, S. T. C. The applica-
tion of immunological methods to the taxonomy of species
within the genus Solanum. Proc. Roy. Soc. Lond. Series B.
151:364—383. 1960.

Goldman, M. Fluorescent Antibody Methods. Academic Press,
N. Y. 1968.

Green, F. The precipitin reaction in relation to grafting.
Genetics 11:73—82. 1926.

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25.

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Hyun, S. K. Serodiagnostic investigation on the affinities of
different species of the genus Quercus and genus Castanea.
Bull. Kyushu Univ. Forests 17:1-60. 1949.

Jensen, W. A. Botanical Histochemistry. W. H. Freeman and Co.,
San Francisco. 1962.

Johnson, M. A. The Precipitin reaction as an index of relation—
ship in the Magnoliaceae. Serol. Mus. Bull. 13:1-15. 1954.

 

Jutila, J. Personal communication. Dept. Botany and Micro-
biology, Montana State University, Bozeman, Montana. 1969.

Kostoff, D. Acquired immunity in plants. Genetics 14:37-77.
1929 O

Kornberg, A. Enzymatic synthesis of DNA. John Wiley and Sons,
Inc. New York, N. Y. 1962.

Redys, J. J., Parzick, A. B. and Borman, E. K. Detection of
group A Streptococci in throat cultures by immunofluorescence.
Public Health Rept. 78:222-226. 1963.

Rives, L. Emploi du serodiagnostic pour la determination de
"l'affinite" des hybrids de vigne. Rev. Vitic, 58:300-302.
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Rives, L. Sur l'emploi du serodiagnoste pour la determination
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Ross, J. G. and Duncan, R. E. Cytological evidence of hybridiza—
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Bull. Torrey Bot. Club 76:414-429. 1949.

 

Sax, K. and Sax, H. J. Chromosome numbers and morphology in the
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Undefriend, S. Fluorescence assay in biology and medicine.
Academic Press, New York. 1962.

APPENDIX A

78
Cytological, Cultural and Histological Studies of the Progressive
Development of Interclonal Juniperus L. Grafts
Introduction

The horticulturally important practice of graftage has, for many
years, been the object of considerable study with major emphasis being
placed on graft compatibility. Many interesting questions have been
raised concerning the developmental biology of the graft union, with
only some of the questions adequately answered. Due to the commercial
importance of fruit growing, most graft studies were on fruit tree
species. The goals of this study were to analyze the influence of
differences in cytology, histology and graft partner vigor upon
ultimate success of the graft union.

Aside from the observation that as botanical relationship between
graft partners becomes increasingly more removed, grafting success
generally decreases; little study has been devoted to the effect of
cytological relationships and their effect on graft success.

A number of cases of sexual "inter-generic" hybrids are known to
occur between genera of the same family. The largest number occurs in
the sub-family Pomoideae of the Rosaceae. These and their known
species are listed in Table l (Cave, 1958—64; Darlington and Wylie,
1955).

Knowledge of the cytogentics of these inter-generic hybrids is
provided principally in two papers by Sax (1929) and Sax and Sax

(1947). They point out that the fact that Sorbus, Aronia, Amelanchier,

 

and Pyrus can be crossed in certain combinations indicates a certain

degree of intergeneric similarity, although the degree of sterility

79

Table 1. "INTERGENERIC'I HYBRIDS IN THE ROSACEOUS SUBFAMILY POMOIDEAE Focke

COTONEASTER = Sorbocotoneaster pozdnjakavii Pojark. 2n=68,85
(X=l7) (S. sibirica x C. melanocarpa) (Gladkova,
1967)

ARONIA————————Sorbaronia Schneid. (X=l7)
(X=17) S, hybrida (Moench) Schneid.
(A. arbutifolia x S. aucuparia)

 

S, fallax (Schneid.) Schneid.
(A. melanocarpa x S. aucuparia)
S, jackii Rehd.
(A. prunifolia x S. americana)
SORBUS S, arsenii (Britt.) G. N. Jones

X=17) (A. prunifolia x S. decora)
. sorbifolia (Poir.) Schneid.
(A. melanocarpa x S. americana)
. alpina (Willd.) Schneid.2n=34(Sax&Sax,1947)
(A. arbutifolia x S. aria)
S, dippelii (Zab.) Schneid.2n=34(Sax&Sax,l947)
(A. melanocarpa x S. aria)

 

lm

AMELANCHIER = Amelasorbus Rehd. (X=17)
(X=l7) A, jackii Rehd. 2n=34 (Sax & Sax, 1947)
(A. alnifolia var. semiintegrifolia
x S. scopulina)

A, hoseri Wroblewski

(A. sp. x S. hybrida?)
A, raciborskiana Wroblewski

(A. asiatica x 8. sp.)

 

 

PYRUS-———-—- Sorbopyrus Schneid.
(X=l7) S, auricularis (KnOOp) Schneid.
(P. communis x S. aria) (3n=51)

 

 

 

CYDONIA Pyronia Veitch
(X=17) S, danieli (Daniel) Rehd.
(C. oblonga x P. communis)
P, veitchii (Trabut) Guillaumin
(C. oblonga x P. communis)

 

 

 

CRATAEGUS ——— = Crataegomespilus Simon—Louis
(X=l7) S, grandiflora (Smith) Bean

MESPILUS (C. oxyacantha x M. germanica)
(X=17) .S. gillotii (Beck) Rehd.

(C. monogyna x M. germanica)
C. dardari Simon-Louis
(C. monogyna x M. germanica)

 

PYRACANTHA Pyracomeles Rehd. ex Guillaumin
(X=l7) ’,///”’//r P, Vilmorinii Rehd. ex Guillaumin
OSTEOMELES (O. subrotunda x P. fortuneana

(X-l7) P. crenat—serrata)

 

 

 

 

80
displayed by the hybrids demonstrate the cytogenetic differentiation
usually found in diverse species. It would be reasonable to suspect
that such sterility might also be correlated with the grafting
success between such species.

Examination of reported interspecific and intergeneric grafts
indicates that in all successful grafts the base chromosome number is
the same for stock and scion. This relationship parallels that found
in sexual hybrids (Table 1).

Dodd and Gershoy (1943) studied interspecific grafts in the genus
.21212, in the interest of establishing a relationship between the
capacity for hybridization and capacity for graft success. They
concluded that no such relationship existed; however, examination of
the results (Table 2) casts some doubt on their interpretation.

Table 2. Results of grafting experiments in the genus Viola.
(from Dodd and Gershoy, 1943)

 

Stocks Scions
V. V. V. V. V.
striata odorata papilionaceae canadensis tricolor
(X=10) (X=10) (X=56) (X=6) (X=13)
V. striata X 0 S 0
(X=10)
V. odorata X 0 S 0
(X=10)
V. papilionacea S S S S
(X=56)
V. canadensis X X X X
(X=6)
V. tricolor 0 X X S
(X-13)

 

X indicates a successful graft. 0 indicates a failure not due to
observed technical difficulties. S indicates a failure which may
be due to technical difficulties.

81

According to the authors, it appeared that insufficient numbers
of grafts were prepared to statistically establish grafting success.
Furthermore they expressed some concern that faulty techniques may
have entered into certain of their failures. It appears from these
data that where base chromosome number of stock and scion deviated
from X=10, greater success occurred where the deviate was used as the
understock.

With consideration for graft component base chromosome numbers,
a re-examination of data obtained by Evans, Watson and Davidson (1961)
suggested that graft compatibility was generally better when such

numbers are the same for stock and scion. Rosa multiflora, a species

 

with 2n=14, and belonging to the subfamily Rosoideae; and Sorbus

aucuparia, Chaenomeles japonica, Malus pumila and Pyracantha coccinea

 

 

 

'Lalandi' all from the subfamily Pomoideae, were chosen for study.
Results of intergeneric grafts among these woody plants are given in
Table 3.

Examination of the above results indicated that greatest success
was obtained when members of the same sub—family were intergrafted.
This may have been due, at least in part, to their all being diploids
with the same absolute and basic chromosome numbers. When members of
different subfamilies with differing basic and absolute chromosome
numbers, were intergrafted, grafting results were highly unfavorable.

The data from Pyracantha have been excluded in arriving at these

 

hypothesis, since the plants were not in a favorable physiological
condition for grafting. The success of self—grafts of the other test

subjects attests to their favorable condition for grafting.

82

Table 3. Survival and average shoot elongation of 4 month old grafts.
(from Evans, Watson and Davidson, 1961)

 

 

 

.EELQE. Understock Percent Success Shoot Elongation
Sorbus Sorbus 100.0 42.3
Malus Sorbus 100.0 39.3
Chaenomeles Sorbus 52.4 19.9
Rosa Sorbus 0.0 0.5
Pyracantha Sorbus 0.0 0.0
Chaenomeles Chaenomeles 85.7 39.0
Malus Chaenomeles 95.2 25.8
Rosa Chaenomeles 0.0 0.0
Pyracantha Chaenomeles 0.0 0.0
Malus Malus 80.0 21.5
Rosa Malus 0.0 0.0
Pyracantha Malus 0.0 0.0
Pyracantha Pyracantha 0.0 0.0
Rosa Rosa 76.2 88.0
Pyracantha Rosa 0.0 0.0

83
In the genus Juniperus, the basic chromosome Number is x=1l, and
all of the species for which chromosome counts have been recorded,

except Juniperus chinensis, are diploids with 2n=22, or have diploid

 

forms (Sax and Sax, 1933; Cave, 1958-1964; Darlington and wylie, 1955).
Juniperus chinensis is reported as being tetraploid, with 2n=44
(Darlington and Wylie, 1955). Van Sloun (1959) evaluated two closely

related North American diploid junipers, Juniperus scepulorum and

 

Juniperus Virginiana, as understocks for four juniper clones——

 

Juniperus chinensis pfitzeriana (an Asiatic tetraploid), Juniperus

 

horizontalis 'Lividus', Juniperus scopuloum 'Montana No. 1' (both

 

 

North American diploids), and Juniperus sabina tamariscifolia (an

Eurasian diploid). Results of this work are given in Table 4.

Table 4. Surviving juniper stock/scion combinations on July 27, 1959.
(from Van Sloun, 1959)

 

Scion/stock combinations % survival

Diploid/diploid grafts 43
Lividus/virginiana 52
Savin/scopulorum 68
Savin/virginiana 43
Montana No. 1/scopulorum 21
Montana No. l/virginiana 13

Tetraploid/diploid grafts

Pfitzer/scopulorum 0
Pfitzer/virginiana 1

 

Van Sloun foUnd no significant difference between Juniperus

scopulorum and Juniperus Virginiana as rootstocks for any given scion.

 

 

From a cytogenetic viewpoint more survival was obtained when scion/

stock combinations involved diploid/diploid grafts than when they

84
involved tetraploid/diploid grafts. This might suggest that grafting
success may be partially attributable to differences in ploidy levels
of stock and scion.
Ahlgren (1962) did work on interspecific pine grafting with a
genus having a base chromosome number of x=12. The species studied

included Pinus peuce, Pinus cembra, Pinus koraiensis (Santamour, 1960),

 

Pinus resinosa and Pinus strobus (Saylor, 1961) all of which are

 

 

diploids with 2n=24. Grafting success for the six combinations studied

are listed in Table 5.

Table 5. Craft survival for six interspecific pine graft combinations.
(from Ahlgren, 1962)

 

Combinations Graft survival
P. peuce/P. strobus 100%
P. koriaensis/P. strobus 83%
P. cembra/P. strobus 77%
P. peuce/P. resinosa 70%
P. koriaensis/P. resinosa 68%
P. cembra/P. resinosa 81%

 

These data substantiate the grafting success when both base and
absolute chromosome numbers of stock and scion are equivalent.

Among the questions arising out of observed incompatibilities in
grafted plants is that of the possible role of biochemical vectors in
control of graft success. In several cases toxic principles which
were transported from one graft partner to another were assumed to be
the causes for graft failures (Toxopeus, 1936; Gur, 1957; Nauriyal

§_t_ a_1. , 1958).

85

Toxopeus (1936) observed that when sweet orange was budded into
sour orange understocks, graft failure was evident in two to three
months with eventual death in 8 to 12 months. Because failures
occurred only when sweet orange was the scion, it was concluded that
a toxic substance was produced by the scion and translocated to the
stock where it was fatal. Similar conclusions were reached by
Nauriyal SE 31' (1958) concerning failures of Eureka lemon scions
grafted upon Trifoliate orange understocks. Because external
symptoms of girdled branches resembled those of the graft combina—
tions they suggested a toxic substance produced in the scion caused
injury to the conducting tissue of the stock.

To explain failures of pears grafted upon quince understock, Our
(1957) proposed that a cyanogenetic glucoside called prunasin, was
produced in the stock phloem adjacent to the union where it decomposed
liberating hydrocyanic acid. He further proposed that the HCN pre-
vented cambial activity and destroyed the conducting tissue near the
union.

Toxic substances in pear on quince grafts have also been postu—
lated by Mosse and Scaramuzzi (1956) as the cause of graft failures.
They suggested that breaks in woody tissue continuity occurred when
these lethal substances accumulated in older phloem near the union
and eventually reached the cambium.

In addition to lethal chemical agents viruses have also been
reported to cause graft failures. In many instances the virus was
shown to be the lethal substance by budding from a susceptible to a

resistant strain, in which case fewer failures were observed.

86

In an effort to explain the dwarfing effect of EM IX understocks
as compared to non—dwarfing EM XVI, Martin and Stahley (1967) studied
the levels of phloridzin (a growth inhibitor), coumaric acid
(decarboxylates IAA), and chlorogenic acid (prevents decarboxylation
of IAA) in the bark of these understocks. Although phloridzin levels
were quite high in both stocks, and could not explain growth con—
trolling behavior, a different mode of metabolism was suggested as the
cause for the differential growth response. Chlorogenic acid was
found to be slightly more concentrated in EM XVI than in IX but
coumaric acid was higher in the latter understock. The total amount
of growth promoting substance, as measured by coleoptile tests, was
higher and growth inhibiting compounds lower in EM XVI than in EM IX.

Studies of graft union structure have concentrated principally
upon the morphological and gross anatomical level. Therefore, certain
questions remain unanswered about stock-scion relationships, many of
which can now be answered with more sophisticated and accurate instru-
mentation.

Tissue discontinuity at the graft union has long been considered
an indicator of incompatible graft unions. Whether this weakness
occurs in the xylem (Waugh, 1904; Bradford and Sitton, 1929a; Chester,
1931; Chang, 1937), or in the phloem (Bradford and Sitton, 1929a;
Bradford and Sitton, 1929b), it is known to result in a structurally
weak graft union (Booth, 1913—1914; Proebsting, 1926, 1928; Chang,
1937). Mosse (1962), pointed out that mechanical weakness may only
be considered a valid criteria for rating compatibility of older trees

since young unions are inherently weak due to lack of lignification of

87
tissue at the union. In fruit tree grafts, apparently normal mature
bearing trees suddenly break at the union leaving a relatively smooth
exposed surface, suggesting that the structural weakness results from
discontinuity of vascular tissue (Hatton, 1936; Mosse, 1960, Mosse,
1962). One example of this has been reported in a self—graft of

Abies concolor (Eames and Cox, 1945) in which no observable cambial

 

union occurred between stock and scion and no parenchymatous tissue
separated the graft components.

In extensive anatomical studies of incompatible graft unions of
pear on apple, Bradford and Sitton (1929b) observed unions in which
a break in cambial continuity occurred, presumably at the end of each
season. This was followed by a re-graft the following spring. The
condition may have resulted from differential growth rates, or
staggered dates of growth cessation at the end of the season. However,
studies on relatively young grafted plants (Sass, 1932; Herrero, 1951;
Evans, Watson and Davidson, 1961), revealed that wood discontinuity of
this type does not occur during the first year after grafting.

Proebsting (1928) described another form of graft abnormality
which is manifested by a distortion of newly formed xylem elements
resulting from uneven growth rates of stock and scion. Such distortion
was not limited to incompatible combinations. Vessel elements in

longitudinal sections of one—year old Sorbus aucuparia self grafts

 

were observed by Evans, Watson and Davidson (1961) in both cross and
longitudinal section at the point of union. This distortion was
believed due to a reorienting of tissue during vascular bridging of

the union.

88

Phloem discontinuity has been recognized as a symptom of graft
incompatibility (Proebsting, 1928; Bradford and Sitton, 1929a;
Herrero, 1951; Lapins, 1959). This symptom may be expressed as
isolated patches of cork between stock and scion (Lapins, 1959), or
as darkly stained, disorganized tissue bounded by rows of cells
resembling the phellem of the periderm (Evans, Watson and Davidson,
1961). It seems plausible that such isolation could occur when
graft partners were in insufficient contact for proper mixing of
tissue. Under these conditions, healing might follow the pattern
known for normal wound healing in which newly differentiated tissue
tends to advance over the wounded surface in a rolling motion
(Bradford and Sitton, 1929b). This would, in a loosely fitted graft,
result in isolation of some periderm tissue.

Phloem discontinuity has indirectly been demonstrated by the
presence of large accumulations of basipetally translocated starch,
at the base of the scion (Booth, 1913-1914; Kostoff, 1928; Proebsting,
1928; Kac, 1931; Roberts, 1934; Chang, 1937; Mosse and Herrero, 1950;
Herrero, 1951). More recently, radioactive tracers have shown block—
age of basipetally translocated phosphorus in scion phloem of inter—
stock grafted apple trees (Dickenson and Samuels, 1956) and in un-
grafted plants in which a ring of bark was deleted from the stem
(Colwell, 1942). In the latter case, accumulated phosphorus was found
in both xylem and phloem at the scion base, suggesting lateral trans—
location at the point of tissue discontinuity.

The possibility exists that the ability of two plants to form a

successful graft union may be controlled to a degree by the inherent

89

similarity or dissimilarity of their anatomy. Such a hypothesis

would seem feasible if one considered that differences, i.e. between
size and/or number of cells of the transport tissue between stock and
scion, could result in a lack of vessel continuity during early stages
of graft healing. This condition could affect early graft survival

as well as exerting a long term effect on plant growth. As an example,
it is known that upward translocation through dwarfing apple rootstocks
is slower than through equivalent tissue of a non-dwarfing understock
(Scholz, 1958). This may be due to the fact that vessel elements of
dwarfing understocks are smaller in cross-sectional area (Beakbane,
1941, 1956; Komarofske, 1947). Matubara (1931) reported a positive
correlation between number of vessels per unit area of stem and graft
survival.

Although investigations of graft union ultrastructure are
relatively new, use of the electron microscope, radio isotopes,
histochemical reagents and other techniques are showing promise as
tools for enlightening our knowledge of cell wall structure.

Buchloh (1960) dealt specifically with cell wall histochemistry
of graft unions. With the electron microscope, he studied the rela—
tionship of lignification to graft compatibility. He reported that
upon contact of stock and scion cells, a middle lamella was formed
between cells of the graft components. If the presently accepted
mode of middle lamella formation is correct, lamella establishment
must occur by atypical means between heterogeneous cells of the graft
union. When a cell undergoes division, the middle lamella forms in

the cell plate, expands laterally to the sidewalls, and is subsequently

9O
overlain by a deposition of secondary wall contributed by both daughter
cells. This mode of pectin deposition would seem improbable under
conditions found in grafted tissue.

Formation of pectic substances between cells of the graft union
may be analogous to that for its formation in outer cell walls of
epidermal cells. Priestly (1943) reported the presence of alternating
lamellar layers of pectin and cutinized cellulose. Because cutin con-
tent of these cellulose lamellae increased in a peripheral direction,
Esau (1960) suggested that this substance may be secreted toward the
surface from epidermal and more deeply seated cells. This explanation
may also apply to the origin of the alternating pectin lamellae, and,
in turn, help to explain middle lamella deposition in graft unions.

The structure of pectic substances has been a subject of some
controversy. Traditionally ruthenium red has been used as a biological
stain for these substances, but it has been shown that this stain is
quite unspecific (Bonner, 1936; Hock, 1942; Kertesz and Loconti, 1944;
Kertesz, 1951). Bonner (1936) explained the lack of specificity as an
affinity for carboxyl groups of the 6th carbon atom, therefore, cellu-
lose, when subjected to mild oxidation, stains with ruthenium red, as
do other compounds having such groups. The degree of specificity is
apparently related to the effect of acidity upon the carboxyl groups
of the pectic substance. Bonner (1936) further pointed out that
ruthenium red does not differentiate between protopectin, pectin,
pectic acid, or calcium pectate.

Basic hydoxylamine has been shown (Cornaz and Deuel, 1954;

Albersheim, Muhlethaler and Frey—Wyssling, 1960) to be more specific

91
for pectin than ruthenium red. It reacts with the esterified carboxyl
groups of pectin to produce hydroxamic acid which forms an insoluble
complex with ferric ions.

The middle lamella is composed primarily of calcium pectate
(Mangin,1888—1893; Bonner, 1936; Kertesz, 1951). However, Ginzburg
(1958, 1961) maintained that cells were cemented together by a gel of
protein and non—cellulosic substances, including pectin. Conversely
Setterfield and Bayley (1957, 1961) suggested that the middle lamella
was a polysaccharide matrix where cellulose was absent rather than a
region of pectate localization. Preston (1964) contended that pectin
was a portion of the cellulose matrix which existed as an adcrusting
substance between the microfibrils. Such a distribution of pectin
could mask the stain reaction of the middle lamella and reduce the
specificity of ruthenium red.

Lignification of cells of the graft union and formation of a
mutual middle lamella between components of pear—quince grafts were
considered as essential to development of structurally sound unions by
Buchloh (1960). He proposed that a secondary chemical differentiation
occurred in the cell wall by which the pectic substance of the middle
lamella disappeared and was gradually replaced by lignin with all
cells but parenchyma subsequently dying. He also pointed out that
compatible grafts contained a lignin concentration in cell walls at
the graft as high as in those some distance away with highest amounts
in the middle lamella.

The present study was undertaken with the following as principal

goals:

92

1. By cultural, anatomical, and histochemical means determine the
success of, and changes occurring during, progressive healing of
successful graft combinations.
2. Determine possible differences in events of objective (1) as they
may occur in interclonal grafts between plants of different ploidy
levels.
3. Ascertain the relative contributions of tissue from stock and scion
to the healing of the graft union.

Materials and Methods

Genetically stable Juniperus chinensis 'Hetzii', Juniperus

 

horizontalis 'Fountain', and Juniperus chinensis 'Pfitzeriana Kallay'

 

 

were used in this study; all are hereafter referred to by their culti—
var names.

On October 24, 1966, the plant material was obtained from a whole—
sale nursery firm£/, and following a cold treatment in a lathhouse,
were potted December 10 in 4 inch clay pots. The plants were then
benched in a 680F. night temperature greenhouse. Scion wood, obtained
from the same source, was stored at 35°F. to break rest and to assure
continued dormancy. Stock and scion wood were approximately one year
from cuttings; stem diameters being 1/8 to 3/16 inches.

Between January 15 and January 17, 1967, the following graft

combinations were prepared using a modified side graft:

Stock Scion
1. J. horizontalis 'Fountain' J. horizontalis 'Fountain'
2. J. chinensis 'Hetzii' J. chinensis 'Hetzii'

l] D. Hill Nursery Co., Dundee, Ill.

93
3. J. chinensis 'Pfitzeriana Kallay' J. chinensis 'Pfitzeriana Kallay'

4. J. chinensis 'Pfitzeriana Kallay' J. chinensis 'Hetzii'

5. J. chinensis 'Hetzii' J. chinensis 'Pfitzeriana Kallay'
6. J. horizontalis 'Fountain' J. chinensis 'Hetzii'

7. J. chinensis 'Hetzii' J. horizontalis 'Fountain'

8. J. horizontalis 'Fountain' J. chinensis 'Pfitzeriana Kallay'

9. J. chinensis 'Pfitzeriana Kallay' J. horizontalis 'Fountain'

The completed grafts were covered with damp Sphagnum moss, and
the entire bench covered with 4 mil clear polyethylene supported on a
wooden frame 24 inches above the plants.

Using standard propagation practices, the understock plants were
cut back to one—half their mass 40 days after grafting; the remainder
being removed at the union 60 days after grafting. Graft survival was
recorded 5 months after grafting.

To study the effect of foreign protein on graft development,

tissue homogenates were made from Juniperus horizontalis 'Fountain',

 

Juniperus chinensis 'Hetzii', and Juniperus chinensis 'Pfitzeriana

 

 

Kallay' leaf and stem tissue. The tissue was prepared by maceration

2/

in a Sorvall Omni Mixer— with enough distilled water to create a
preparation of pasty consistancy. On January 17, 1967, the following
treatments were prepared by applying the tissue homogenate to the graft

incision prior to completion of the graft:

Graft Combination Extract Used

 

 

1. 'Fountain' on 'Fountain' 'Hetzii'

g/Ivan Sorvall, Inc., Norwalk, Conn.

94

2. 'Fountain' on 'Fountain' 'Pfitzeriana Kallay'
3. 'Hetzii' on 'Hetzii' 'Fountain'

4. 'Hetzii' on 'Hetzii' 'Pfitzeriana Kallay'
5. 'Pfitzeriana Kallay' on 'Pfitzeriana Kallay' 'Fountain'

6. 'Pfitzeriana Kallay' on 'Pfitzeriana Kallay' 'Hetzii'

Five months after grafting, survival data was recorded.

At six 10 day intervals following grafting, 3/4 inch stem segments
containing the graft union were collected for each combination. Upon
harvest, the graft unions were immediately frozen in polyethylene bags
to await sectioning.

Samples were evacuated and infiltrated for one hour in a 1.5%
solution of Reteng/ using a venturi type vaccuum aspirator. Sections
were cut at 10 microns using a refrigerated microtome (Cryostat)£/
with a cutting temperature of -150C. Sections were picked up directly
from the microtome blade on glass slides coated with a thin film of
0.75% Reten.

Pectin distribution, especially in the middle lamella was studied
on a series of preparations from all graft combinations at 10, 20, 30,
40, 50, and 60 days after grafting. The sections were stained with
ruthenium red (1: 5000 aqueous solution) for 12 hours at room tempera—
ture and the cover slip mounted with glycerine jelly.

To investigate tissue lignification in the region of the graft

union, a series of sections from all combinations and dates were

stained with Azure B for one hour at room temperature. This was a

E/A cationic, water soluble polymer; Hercules Powder Co.

fi/International Equipment Co. (Model CTD)

95

slight modification of the procedure suggested by Jensen (1962).

Following Jensen (1962), cell walls were studied by a stepwise
extraction of each component after which sections were stained with
the non-specific stain, periodic acid-Schiff's reagent. The sections
were stained for 30 minutes in periodic acid followed by 15 minutes in
Schiff's reagent. Color photomicrographs of the stained sections were
prepared and compared for reduction in stain intensity; a measure of
the amount of each cell wall constituent removed at each extraction.

Wilson (1961) reported difficulty in maintaining adhesion of
woody plant sections to microslides during extraction in strong base.
He was able, however, to overcome the problem during cell wall extrac-
tion by using a thin layer of fiber glass over the sections. This was
secured by a second microslide held to the first by aluminum wire to
form a "sandwich".

This did not appear feasible in this study since the fiber glass
and wiring might disrupt the fragile callus tissue of the graft union.
For this reason, several combinations of materials were used to over—
come the loss of sections during extraction. One combination was a
piece of green saran shade cloth which was overlain with % inch hard-
ware cloth and secured with paper clips. Another combination used
saran shade cloth overlain with fine stainless steel mesh which was
held together with hair clips.

All stained preparations, with the exception of those stained with
ruthenium red were mounted permanently in Permount§/, and stored until

microscopic examination.

E/Fisher Chemical Co.

96

For anatomical study, sections were stained with safranin (15
minutes) and counterstained with aniline blue (1 minute); a stain
combination considered to be ideal for gymnosperms (Johansen, 1940;
Jensen, 1962). The staining procedure was altered from that described
by Jensen (1962) by the inclusion of a stepped alcohol dehydration
series between safranin and aniline blue. Poor preparations resulted
when slides entered the clove oil-absolute alcohol-xylene solution
unless this was done.

The possible correlation between anatomical differences of stock
and scion and graft success was studied using an eyepiece micrometer
to determine mean cell size for vessel element and ray cell diameters
and lengths in ungrafted wood of each clone. Data on diameter of
newly formed, wound induced vessel elements found between faces of the
graft incision was also recorded. Newly formed vessel elements were
measured in 60 day old grafts only.

As a measure of relative vigor, counts of normal tracheids and
those stimulated by wounding during grafting and produced subsequently,
were made for all combinations of junipers studied and at the six
dates following grafting.

Figure 1 serves to illustrate sectors of the grafted plant here—
after referred to as "Normal" tracheids and ”Wound" tracheids in

reference to cell count and cell measurement data.

Results and Discussion
The graft survival data (Table 6) revealed that general graft

success was 58% to 100% for all graft combinations at the end of

97

Understock
"Normal" Understock Tracheids Wound Tracheids
(produced since grafting) (produced since
grafting)

 

Phloem and
Cortex

5 .‘ 1“" 9 f".
. Egtdztzzgfi
52‘ ‘ ‘ ‘1‘“.—

053¢=Efi: 55-;L"

Scion

Wound Tracheids
”Normal" Scion Tracheids (produced since
(produced since grafting) grafting)

Figure 1. Diagramatic representation-of a typical 60 day old graft
union viewed in cross—section. Points are indicated at
which cell counts and cell measurement data were taken.

98

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99

5 months. Self-grafted control plants, the diploid and triploid

junipers, Juniperus horizontalis 'Fountain' (2n=22) and Juniperus

 

chinensis 'Hetzii' (3n=33) (Section 1, Thesis) were highly successful
while an extremely low success was observed for Juniperus chinensis
'Pfitzeriana Kallay' (4n=44). The success Obtained on 'Fountain'

and 'Hetzii' junipers signified that adequate grafting techniques were
used for these clones.

Intergeneric grafts fell into two distinct groups. The most
successful combinations were 'Hetzii' on 'Fountain', 'Fountain' on
'Hetzii' and 'Fountain' on 'Pfitzeriana Kallay', in which all 12 plants
survived. Least successful combinations were 'Hetzii' on 'Pfitzeriana
Kallay', 'Pfitzeriana Kallay' on 'Hetzii' and 'Pfitzeriana Kallay'
on 'Fountain'.

Almost all highly successful grafts contained 'Fountain' as a
component, conversely, all less successful grafts had 'Pfitzeriana
Kallay' as one partner. This suggested the possibility that 'Fountain'
may have exerted a stimulating effect on graft healing while
'Pfitzeriana Kallay' suppressed or failed to contribute to graft union
development. Such a condition might arise from insufficient growth
factors or to the presence of inhibitors. This supposition is sup—
ported by the 1ow success observed for self grafts of Juniperus
chinensis 'Pfitzeriana Kallay' and by its poor performance observed
when used as a scion for Juniperus Virginiana by Van Sloun (1959).
'Hetzii' being involved in a large number of successful grafts,
suggests that it may exert a beneficial influence upon graft union

formation.

100

Results of preliminary studies using tissue extract treated self-
grafts suggested pursual of this research to be desirable. Because
extract treated grafts were found to respond differently from those
of non—treated controls it appeared that applied extracts produced a
marked influence upon graft success (Table 7).

Application of 'Hetzii' or 'Pfitzeriana Kallay' extract to grafted
surfaces of 'Fountain' juniper resulted in no apparent effect upon
graft success, but reduced scion vigor was observed when 'Pfitzeriana
Kallay' extract was used and increased vigor with the former extract.

Extracts of 'Fountain' when applied to 'Pfitzeriana Kallay'
grafts resulted in a significant increase in graft success. Whereas
untreated self-grafts of 'Pfitzeriana Kallay' (Table 6) were only 25%
successful, these grafts with extract applied were 80% successful. A
marked increase in vigor accompanied the observed grafting success
(Figure 2).

These results suggest the presence of a growth factor in Juniperus

horizontalis 'Fountain', which when applied to the graft union stimu-

 

lates healing of the 'Pfitzeriana Kallay' graft union. This hypothesis
is supported by the results of the previous experiment, where in most
successful grafts, 'Fountain' juniper was a component. The ease of
rooting add circumstantial evidence of the presence of such a substance.
Lanphear and Meahl (1966) reported 100% rooting of Juniperus
.hggizgntaliadplumgsa under growth chamber conditions which also

supports this hypothesis. Chromatograms of tissue extracts from this

juniper yielded a substance (rf=0.8) which exhibited strong root

producing capacity in the mung bean bio assay.

101

 

 

Figure 2 - The effect of application of leaf and stem extract to
graft surfaces of Juniperus chinensis 'Pfitzeriana
Kallay' self grafts. No extract (center), 'Fountain'
extract (right), and 'Hetzii' extract (left).

102

NH

NH

NH

HH

HH

no\MH\o

fiance

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q q q
N q N
N m N
q q q
q q q
m q q
o o m
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HHH .mmm HH .mmm H .mmm

 

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.%mHHmM mamHumuunm. .5
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.HHNumm. mHmaoaHso .h
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.GHMuasom. mHHMuaomHuon .h
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meHumaHnaoo

.o magma

103

Earlier work (Section 2 of thesis) indicated that initiation of
callus tissue production in self grafts of Juniperus chinensis
'Pfitzeriana Kallay' was 20 days behind the other junipers studied.
Speculatively 'Fountain' juniper may stimulate tissue production in
the 'Pfitzeriana Kallay' graft union. The necessity of callus tissue
production to grafting success has been well established among propa-
gators, and was reported by Harmon and Weinberger (1967) to correlate
positively with successful grafting of grapes.

Extract of 'Hetzii' on 'Pfitzeriana Kallay' self grafts resulted
in no observable difference in graft success when compared to untreated
controls, and ultimate vigor was less pronounced than when 'Fountain'
was the extract source. This observation might suggest a lack, in
'Hetzii', of the beneficial substance possessed by 'Fountain'.

Because 'Hetzii' self grafts were highly successful in control
grafts, it was difficult to explain the poor results obtained when
'Pfitzeriana Kallay' extracts were applied to these graft unions.
Although observed vigor was equivalent to control grafts, total
graft success was markedly suppressed by application of 'Pfitzeriana
Kallay' extract. One might speculate that, because it is quite un-
successful as a self graft, extracts of 'Pfitzeriana Kallay' may
interfere with and have a suppressive effect upon the 'Hetzii' self
grafts. Although a high degree of success was obtained when
'Pfitzeriana Kallay' extract was applied to 'Fountain', some reduc-
tion of growth was noted. In this case, any suppression of graft

success by 'Pfitzeriana Kallay' may have been countered by the

opposing effect of the 'Fountain' growth factor.

104

The reason for failure with 'Fountain' self grafts treated with
'Hetzii' is unknown.

Comparative measurements of ray cells and tracheids produced some
interesting relationships which may help explain structurally some of
the differential responses in interclonial grafts of Juniperus

horizontalis 'Fountain', Juniperus chinensis 'Hetzii' and Juniperus

 

 

chinensis 'Pfitzeriana Kallay'. Cell measurements are presented in
Table 8.

Ray cell dimensions revealed significant differences between
'Pfitzeriana Kallay', 'Fountain' and 'Hetzii' in length and cross-
sectional area; the latter two not differing significantly from each
other.

Although significant differences did not exist between all means,

a direct relationship was noted between ray cell measurement and ploidy
level of the junipers studied ('Fountain' 2n=22, 'Hetzii' 3n=33, and
'Pfitzeriana Kallay' 4n=44). This relationship was positive for ray
cell tangential diameter and negative for ray cell length.
'Pfitzeriana Kallay' ray cells were significantly shorter and thicker
than those of the other taxa. Therefore, this juniper should possess
a greater number of tangential walls per increment of radial distance;
a condition which might influence lateral translocation in the plant.
Esau (1953) points out that ray cell length increases with plant vigor.

Ray cell dimensions observed by Bailey (1920) in one year old

stems of Pinus strobus differed markedly with similar measurements for

 

juniper. Measurement of 50 ray cell initials revealed a length of

17.8 microns with a tangential height of 22.9 microns and a tangential

105

.uamummeU hHuamoHMchHm nos mumuumH wXHH kn woBOHHow mammz\fl

mqw.m nqw.~ mmH.m mammz
NON.wN Nom.om Nom.mm .>.o
.deunsom. .HHnumm. .zmHHmM.

 

AmaouoHEV Mooum map mo GonHoGH “mayo map um voodvoum mmeEmHm meSUmMH wofiuom kHBma mo

 

 

 

 

 

mmo.mON nmm.qqm mmm.mHN mam.m mam.m mo~.m
NQH.wH Noo.mH Nom.ON NNo.ON Noq.mH Noq.HN
.GHMuasom. .HHNumm. .%MHHmM. .aHmuasom. .HHNuom. .hmHHmM.
AmaouoHEv suwqu vagomuH AmaouoHav HoumamHm mesomuH
nmm.om noq.om qu.HN nNm.m Amm.© mHm.o
Noq.mN Nom.mH Now.mH Nom.mH Nom.HH Nom.mH
.chuasom. .HHNumm. .%mHHmM. .deuasom. _HHNumm. .%MHHmM.
AmaouoHev amwqu Hmvam HHmo mum AmaouoHav HmumSMHQ HmHuauwama HHmo Mom

.mfioum pHOIHmoklosu Eoum mumv How momma mwamu mHaHuHse
m.amossm paw .%uHHHanum> mo muaoHonmmoo .Aomnav muamaoudmmoa HHoo hm“ can menomuu ammz

muoumEmHm

.w mHLMH

106

width of 13.8 microns. All but the latter measurement was larger than
that observed for junipers. Because ray initials are typically flat-
tened in a radial direction, tangential width in the pine would be
expected to exceed that observed for Juniperus L. for mature cambial
derivitives.

No significant difference in tracheid diameters was observed.
However, 'Hetzii' had a significantly greater tracheid length.

Even though no differences were detected among clones with respect
to mature tracheid diameter, 'Hetzii' differed significantly from the
other junipers in diameter of new tracheids in the graft incision.
Coefficients of variation for these means indicated a high degree of
variability when compared to the other cells.

Tracheid length of 'Fountain' and 'Pfitzeriana Kallay' did not
differ significantly, but both had shorter tracheids than 'Hetzii'.
Measurements were considerably smaller than those reported for

tracheids of the arborescent Juniperus Virginiana reported by Bailey

 

and Tupper (1918).

The size of new tracheids produced in the graft union were two
or three times the diameter of those produced by non—wounded tissue;
an observation also reported by Block (1952) in wound meristems, thus
suggesting that graft union formation is basically a form of wound
healing.

The comparative vigor of the nine interclonal juniper grafts and
their component parts during the 60 days following grafting was
measured by cell counts of all new differentiated tracheids arising

during that period. The raw data for these counts are given in

107

Appendix Tables B1 and B2 respectively and condensed values are given
in Tables 9 and 10 of the text.

Analyses of variance for wound tracheids and normal tracheids
were computed independently on the assumption that no interaction
existed between cell counts of each. When plotted (Figure 3) the
parallelism between curves for the two classes of cell counts verified
that interaction was absent.

Concommitantly this plot indicates that cell production in the
stock and scion are similar. The slight depression in scion cell
production in 30 day old normal wood was due to a general cessation
of growth during the period when greater metabolic activity was
occurring at sites of injury. Because wound tissue cell size exceeded
that of normal cambial derivitives (Table 8), supression in normal wood
production was not alleviated by an equal increase in cell numbers in
the injured scion wood.

A significant F value for date means of wound tracheids and normal
tracheids (Tables 9 and 10) signified that cell production at each date
differed statistically from other dates.

The mean cell counts for all graft combinations revealed that

fewer cell numbers were observed in Juniperus horizontalis 'FOuntain'

 

and Juniperus chinensis 'Pfitzeriana Kallay' self grafts in both normal

 

and wound induced tracheids (Tables 9 and 10). The greater number of

normal cells suggested that Juniperus chinensis 'Hetzii' self grafts

 

were the most.vigorous of the clones studied. If 'Hetzii', a triploid
juniper (section I of thesis), originated from diploid and tetraploid

parents; on the basis of hybrid vigor, it might be expected to produce

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108

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110
a greater number of cells than either 'Fountain' (2n=22) or
'Pfitzeriana Kallay' (4n=44).

Cell counts showed 'Hetzii' on 'Pfitzeriana Kallay' grafts to
have the greatest vigor in normal and wound induced tissue. 'Fountain'
or 'Kallay' on the other understocks produced statistically equivalent
numbers of cells in injured wood but all values were significantly
greater than their self grafts. In normal wood 'Hetzii' self grafts,
'Kallay' on 'Hetzii' and 'Fountain' on 'Kallay' all responded equally
but with significantly larger numbers of cells, than the other self
grafts, 'Fountain' on 'Hetzii', or its reciprocal.

In both wounded and normal wood, 'Fountain' on 'Hetzii' grafts
produced a significantly greater number of cells than their reciprocals
which may be due to the differences in vigor of the self grafts of
these clones. A comparison of the production of 'Fountain' versus
'Kallay' on 'Hetzii' in normal wood revealed significantly higher
values for 'Kallay' than for 'Fountain'. Thus, the influence of stock
on scion may be related to ploidy level. Wound tracheid numbers were
not significantly different.

Significant differences among graft partners of both classes of
wood (Tables 9 and 10) was interpreted as representing the differences
between grand stock and scion means with the scions producing less
cells than understocks. This relationship is illustrated in Figure 3.

The interaction between combinations and dates was found to be
significant for both wound and normal tracheids. This relationship
may have resulted from the failure of each graft combination to produce

like numbers of cell at any given date. The most obvious example of

lll

 

 

 

    
 

L stocks
‘ NORMAL TRACHEIDS
scions

U)
4.)

a

a

o
L)
H
r-l

o
L)

20 5o 46 50 30
Days after grafting
15 4,
stocks
.L WOUND TRACHEIDS
a scions
I
10

0)

J.)

a

s

o

t)

H

H4

8

5

 

 

20 3’0 40 5'0 6 0
Days after grafting

Figure 3 — Mean cell counts at wounded and un-wounded sites of stocks and
scions of interclonal juniper grafts at five dates after
grafting.

112
this interaction was the lack of cell division in 'Kallay' self grafts
during the initial 30 days after grafting. By 40 days, however, these
grafts were producing nearly equivalent numbers of cells. A similar
but not as dramatic lag was observed in 'Fountain' self grafts in
both wounded and normal wood during the same period.

'Hetzii' self grafts also exemplified the date by combination
interaction. It was noted that in normal wood, cell counts at 50 and
60 days were equivalent, thus indicating a leveling off of cell divi—
sion by that time. The wound induced tracheids of this graft had a
similar plateau between 40 and 50 days after which activity was
resumed to 60 days. A leveling off of cell production in wounded
tissue was also noted in 'Kallay' on 'Hetzii' grafts between 40 and
50 days following grafting, but this behavior was not clearly evident
in normal wood.

As a result of a failure of graft partners to respond in like
fashion at any given date, a significant interaction was detected
between partners and dates. Contributing to this interaction was the
fact (Figure 3) that scions, in both wound induced and normal wood,
produced a quantity of cells at 50 days equal to that produced by
stocks at 40 days. These dates also show cell production to be equal
in 20 day old grafts in both wounded and un—wounded wood; an observa-
tion interpreted as a lack of new tracheid production by either graft
component. The convergence of the lines in Figure 3 at 60 days signi—
fied that healing and associated balance of cell production was
nearing completion.

The significant interaction observed between graft combinations

113

and graft partners (Table 9 and 10) appeared to result from a failure
of each clone to respond equally well when used as a stock and when
used as a scion for the other clones. These relationships are pre-
sented in Table 11.

In combinations of 'Fountain' on 'Kallay' significantly greater
numbers of cells were produced in normal wood of both stock and scion
than in other combinations with 'Fountain' as a scion. When recipro—
cal graft means were examined for normal tissue, the same relationship
held. This observation indicated the enhancing effect of 'Fountain'
on 'Kallay' self grafts in the plant extract experiment results
reported earlier (Table 7).

When wound tracheid numbers were compared with 'Fountain' as the
scion, response was greater in 'Fountain' on 'Hetzii' than in other
combinations; this being considered a probable reflection of the
effect of 'Hetzii's' greater vigor on the low vigored 'Fountain'.

The influence of 'Fountain' scions on 'Kallay' stocks was again
apparent when stock counts were made. As an understock, 'Fountain'
also had a significant influence upon cell counts of 'Kallay' scions,
but no significant differences were observable between 'Fountain'
understocks when using 'Hetzii' and 'Kallay' as scions.

Examination of data for 'Hetzii' scions indicated that in both
wound induced and normal wood, cell production increased with
increasing ploidy level of the stock in the order: 'Fountain',
'Hetzii' and 'Kallay'. This relationship held for understock cell
production for wound tracheids only, the 'Hetzii' stock values were

significantly greater than those for 'Kallay' in normal wood. As an

114

Table 11 - Comparative mean radial cell counts for wound induced and

normal stock and scion tissue produced after grafting in

nine interclonal juniper graft combinations.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

N 0 R M A L T R A C H E I D S
cion 'Fountain' 'Hetzii' 'Kallay' StOCk scion
stock means means
5.7 7.7 10.9 8.1
'Fountain' \ \
8.8 11.0 13.0 10.9
8.0 10.0 9.3 p 9.1
'Hetzii' \
12.1 12. 13.2 12.6
8.1 12.9 5.7 8.9
'Kallay' \
13.7 11.3 8.2 11.1
scion
means 7.3 10. 8.6 8.7
stock
means 11.5 11.6 11.5 11.5
W O U N D T R A C H E I D S
scion 'Fountain' 'Hetzii' 'Kallay' StOCk scion
stock means means
3.9 ' 4.3 6.9 5.0
'Fountain' ‘\\\\\“\\\\
4.3 6.7 7.0 6.0
6.9 5.2 5.5 5.9
'Hetzii' \ \
7.3 7.1 8.1 7.5
5.6 7.6 3.7 5.6
'Kallay'
8.7 9.5 6.2 8.1
scion
means 5.5 5. 5.5
stock
means 6.8 7.8 7.1 7.2

 

 

 

115

understock, 'Hetzii' did not follow the pattern observed for scions.
Although significantly greater numbers of cells were produced with
'Kallay' as the scion in both classes of wood, self grafts exceeded
'Fountain' in cell production; the reverse was observed for wound
tracheids.

In contrast to the generally observed tendency for scion cell
production to increase directly with ploidy level of the understock,
'Kallay' scions varied inversely with ploidy level; largest counts
being observed where 'Fountain' was the understock. In understocks
this trend was broken by the larger numbers of cells produced by
'Hetzii' understocks. In reciprocal grafts 'Hetzii' scions also
produced the largest cell numbers when combined with 'Hetzii' scions,
while in normal wood, it produced significantly more cells when
'Fountain' was the scion.

The major problem in adapting the cell extraction procedure to
graft unions was the maintenance of the structural integrity of the
grafted tissue throughout the extraction procedure. A procedure
developed in this study was satisfactory for securing the tissues to
the slide, however, some of the parenchymatous callus was still lost
during extraction. The use of silicone rubber to adhere the sections,
as described by Holmes and Brown (1967), appeared to offer promise
for overcoming problems encountered, but this was published after the
work was completed.

To study pectic substances in common walls of mixed stock and
scion cells, a series of sections from graft combinations were stained

with ruthenium red.

116

Although reported to lack specificity for pectins (Hock, 1942;
Kertesz and Loconti, 1944; Kertesz, 1951; Bonner, 1936), ruthenium
red clearly delineated the middle lamellas of mature tracheids. In
callus cells, however, the stain was diffused throughout the entire
cell wall and masked the middle lamella. This precluded the possi-
bility of distinguishing stock callus from scion callus on the basis
of presence or absence of middle lamella.

Localization of ruthenium red in typical graft union tissue in
longitudinal section is illustrated in Figure 4A. The lack of a
clearly defined middle lamella is shown in the photomicrograph. The
lack of definition may be an indication that this structure is com-
prised of other substances as well as calcium pectate. If composed
of a protein gel (Ginzburg, 1958, 1961), or cellulose adcrusted with
pectin (Preston, 1964) its presence might be quite ill defined.

The pectin localization in longitudinal section of wood ray cells
at the xylem-pith junction is illustrated in Figure 4B. Heavy pectin
localization was evident in the tangential walls (arrows) when com—
pared to the staining of the radial cell walls. Correspondingly

there were more pits in the tangential walls.

 

Figure 4 — Pectic substance localization in juniper graft unions
and in medullary ray cell walls. A. The diffuse nature
of ruthenium red staining substances in graft callus
cells. B. Typically heavy concentration of pectic
substance in tangential walls of ray cells and charac—
teristic heavy pitting in these walls.

 

118

Summary

Interploidy grafts of Juniperus horizontalis 'Fountain' (2n=22),

 

Juniperus chinensis 'Hetzii' (3n=33), and Juniperus chinensis

 

 

'Pfitzeriana Kallay', were prepared to study relationships between
ploidy level and graft success. Self grafts of the diploid and tri—
ploid junipers were highly successful while those of the tetraploid

Juniperus chinensis 'Pfitzeriana Kallay' were unsuccessful. Since

 

most unsuccessful graft combinations contained 'Pfitzeriana Kallay'
as one partner it was postulated that an insufficient level of a
growth substance or growth inhibitor may have been responsible.

Most successful interclonal grafts contained the diploid juniper

Juniperus horizontalis 'Fountain', thus suggesting the presence of a

 

substance capable of improving graft union development.

Self grafts were prepared and tissue extracts from one of the
other junipers was applied to the cut surfaces in an attempt to detect
species limited substances which might influence graft union develop—
ment. The effect of plant extracts upon graft formation and develop—
ment was an alteration of the percentage of graft successes in the
difficult to graft combinations.

Application of 'Hetzii' or 'Pfitzeriana Kallay' extract to the
cut surfaces of the 'Fountain' juniper produced no changes in total
success, but 'Pfitzeriana Kallay' extract produced less visible
scion vigor.

The most marked effect of tissue extract on graft success was the

application of 'Fountain' extract to cut surfaces of 'Pfitzeriana

Kallay'. This treatment resulted in 80% grafting success as compared

119
to 25% when un-treated. Ultimate vigor of this treatment was superior
to self grafted controls and the other treated grafts.

The presence of a growth stimulating substance in Juniperus
horizontalis 'Fountain' was postulated, and absence of, or reduced
levels in Juniperus chinensis 'Pfitzeriana Kallay' were suggested for
the differential response of the extracts on graft success and vigor.

The 20 day delay in callus tissue production in self grafts of
'Pfitzeriana Kallay' as compared to 'Hetzii' or 'Fountain' suggests
that the failure and lack of scion vigor in grafts involving
'Pfitzeriana Kallay' may result from this condition.

Self grafts of Juniperus chinensis 'Hetzii' responded very poorly
when treated with 'Pfitzeriana Kallay' extract. This suggests that
this extract may interfere with normal callus production and graft,
development. The possible suppressing effect of this extract and the
total lack of success of 'Fountain' treated 'Hetzii' self grafts
were impossible to explain and await further study.

Results of ray cell measurements revealed that the cells of
'Pfitzeriana Kallay' were significantly shorter and larger in diameter
than those of the other two junipers. No statistical difference was
observed between 'Hetzii' and 'Fountain' junipers. However, the
tracheid cells of 'Hetzii' were significantly longer. Diameter of
'Hetzii' wound tracheids was significantly smaller than those of
'Pfitzeriana Kallay' or 'Fountain', with no difference between
'Pfitzeriana Kallay' and 'Fountain'. Wound tracheid cells were two
to three times the diameter of normal mature cells.

Cell counts of new tracheids, both in normal wood and in tissue

120
arising at graft surfaces, revealed that the contribution of the stock
was greater than the scions. The lowest numbers of cells were in the

self grafts of Juniperus horizontalis 'Fountain' and Juniperus

 

chinensis 'Pfitzeriana Kallay'. The most vigorous combination was
'Hetzii' on 'Pfitzeriana Kallay'. The highest cell counts among self
grafts were in 'Hetzii'. This may be a possible ”hybrid vigor" of this
triploid juniper over the diploid 'Fountain' and tetraploid
'Pfitzeriana Kallay'.

In both wound induced and normal tissue, 'Pfitzeriana Kallay' self
grafts failed to initiate new tracheal cells during the first 30 days
after grafting. A similar but shorter lag in cell production was found
in 'Fountain' self grafts. In normal wood of 'Hetzii' self grafts a
plateau in cell production was reached between 50 and 60 days, while in
wounded wood, the plateau was reached between 40 and 50 days. The fact
that stock and scion cell counts had approximated one another by the
60th day following grafting was considered to be indication that graft
healing was near completion by that date.

With the exception of 'Fountain' on 'Hetzii' grafts, when used as
scions, cell counts of these clones increased significantly with
increasing ploidy level of the understock on which they were grafted.
With 'Pfitzeriana Kallay' as a scion, however, the inverse relationship
was observed.

Of the three clones used as scions, highest mean cell counts were
observed with 'Hetzii', but as an understock, greater counts were
observed in 'Pfitzeriana Kallay'. 'Hetzii' scions produced the largest

cell numbers in normal wood while 'Pfitzeriana Kallay' numbers were

121
greater in wounded wood.

In all grafts involving 'Fountain' and 'Pfitzeriana Kallay', cell
counts of 'Pfitzeriana Kallay' were significantly higher than the self
grafts of each clone. This substantiated the effect observed when
tissue extracts of 'Fountain' juniper was applied to 'Pfitzeriana
Kallay' resulting in increased graft success.

Due to heterogeneity and structural weakness of the tissue, a
cell wall extraction procedure for studying cell wall constituents
during graft healing was not successfully adapted to this investiga—
tion.

Staining with ruthenium red was found to be rather diffuse
throughout primary cell walls rather than being limited to a well
defined lamellar area thus eliminating this as an identification of
cellular contribution by the stock and scion. Observations were
made, however, on the highly pectinaceous nature and heavy pitting of
xylem ray cell tangential walls as compared to their radial walls.

Of greatest significance among results of this study was the
effect of certain juniper clones on grafting success. The beneficial

effect of Juniperus horizontalis 'Fountain' upon graft success and

 

cell production of Juniperus chinensis 'Pfitzeriana Kallay', whether

 

applied as an extract to self grafts or as a graft component, was a
prime example. Such findings justify further investigation into the

nature of the effects observed in the present study.

122

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127

Summary

Using Juniperus horizontalis 'Plumosa Compacta', Juniperus

 

horizontalis 'Fountain', Juniperus sabina 'Von Ehron', Juniperus

 

 

chinensis 'Hetzii', and Juniperus chinensis 'Pfitzeriana Kallay',

 

a series of related experiments were conducted to study the cyto-
logical, anatomical, nutritional, immunological, and cultural aspects
of self and interclonal graft unions during their development.
Because of possible cytological implications in graft union
behavior, somatic chromosome counts were made for three previously
uncounted juniper clones. An unexpected chromosome number of 3n=33

was obtained for Juniperus chinensis 'Hetzii', and counts of 2n=22

 

were noted for Juniperus horizontalis 'Plumosa Compacta' and

 

Juniperus sabina 'Von Ehron'.

 

Comparative anatomical studies of Juniperus horizontalis

 

'Fountain', Juniperus chinensis 'Hetzii', and Juniperus chinensis

 

'Pfitzeriana Kallay' revealed that tangential medullary ray cell
diameter increased with increasing ploidy level.

Anatomical study of interclonal juniper grafts showed no basic
difference in developmental sequence; only differences in timing of
each stage.

Filling of voids between stock and scion with callus from phloem
and cortex was the first observable event in healing. This stage was
reached by the end of 20 days and was first observed in grafts with
'Fountain' as a component, and last in those containing 'Pfitzeriana

Kallay'.

128

After 20 to 30 days, isodiametric cells appeared from uninjured
cambial tissue adjacent to the graft incision, which by tangential
and radial divisions overwalled the injured graft surfaces. This did
not occur in 'Pfitzeriana Kallay' until the period 30 to 40 days after
grafting.

By radial divisions, overwalling cells filled the area between
stock and scion with well organized tissue and crushed intervening
callus by the end of 40 to 50 days.

In 50 to 60 day old grafts, a "cambial bridge", composed of
typical elongated tracheids, formed between cambial rings of stock
and scion. From the new cambial bridge, new xylem began to form.

By this date, new tissue contribution was equivalent from stock and
scion.

Study of mixed graft tissue revealed that common walls of
heterogeneous cell pairs appeared as double wall structures, each
with middle lamellas. Abnormally large numbers of pits, and occa-
sional blind pits, believed due to unaligned pit pairs, were also
observed.

Mineral distribution of interclonal juniper grafts, studied by
ashing of entire graft components, showed 'Hetzii' scions to accumu-
lated more calcium and potassium than in understocks. 'Plumosa
Compacta' self grafts accumulated potassium in the understock while,
in other self grafts, this element was equally distributed.

Data obtained from ashing and photometry indicated that rate of
translocation of elements studied was, in decreasing order, potassium,

calcium, and magnesium; the latter was relatively unrestricted at the

 

 

129

graft union.

Electron microprobe X-ray analysis showed that calcium tended to
accumulate in the understock xylem of typical interclonal juniper
grafts, while potassium and magnesium appeared to move readily across
the graft union.

Although elements were equally distributed between phloem and
cortex of stock and scion, slight magnesium accumulated was found in
the phloem and significant amounts of calcium were observed in the
scion cortex. Accumulation of this element in understock xylem led to
a postulated lateral movement to the understock cortex where diffusion
through the union occurred.

Microprobe analysis indicated the rate of element translocation
at the union to be, in decreasing order; magnesium, potassium and
calcium.

To determine the feasibility of serological techniques for graft
union study, interclonal graft union microslide preparations were
treated with fluorescein labelled antibody preparations. Immunization
of inbred white mice with leaf and stem extract of Juniperus

horizontalis 'Fountain' and Juniperus chinensis 'Pfitzeriana Kallay'

 

 

resulted in strong, titer levels as measured by microprecipitin reac—
tions. Cross reactions showed 'Fountain' to have greatest antisera
specificity.

In 'Fountain' on 'Pfitzeriana Kallay' grafts incubated with
'Fountain' antibody, fluorescence of the scion exceeded that of the
stock. Labelling with 'Pfitzeriana Kallay' antibody resulted in major

fluorescence in the stock.

130

Though antibody specificity was generally greatest in parenchyma-
tous tissue, 'Fountain' antibody fluoresced more in the xylem.

During the course of serological study, micro Kjeldahl determina-
tions showed 'Pfitzeriana Kallay', a tetraploid with 4n=44, to contain
slightly more than twice the amount of protein nitrogen than the
diploid 'Fountain' (2n=22); suggesting that differences in fluore—
scence levels may have been partly due to amount of protein available
for labelling.

Among the self grafts, 'Hetzii' and 'Fountain' were found to be

successful, while 'Pfitzeriana Kallay' was unsuccessful. Degree of

 

success was accordingly affected in interclonal grafts involving each
of these clones as components.

Studies with self grafts in which the cut surfaces had been
treated with a leaf extract from another clone at time of grafting
showed 'Fountain' to improve graft success of 'Pfitzeriana Kallay';
self grafts of which were not successful in control grafts.

When applied to 'Fountain' self grafts, 'Pfitzeriana Kallay'
caused no change in graft success but a reduction in scion vigor was
noted. 'Hetzii' self grafts, however, responded very poorly to
application of this extract.

Cell measurements indicated 'Pfitzeriana Kallay' to have the
shortest, thickest ray cells of the junipers studied, while 'Hetzii'
tracheids were found to be the longest. Tracheids formed subsequent
to wounding were larger than those in normal tissue.

Cell counts revealed that greatest contribution of new tracheids

arose from the understock of juniper grafts. Among self grafts,

 

131
'Hetzii' scions produced largest numbers of cells while 'Pfitzeriana
Kallay', used as a scion for interclonal grafts, produced greatest
cell numbers.

'Pfitzeriana Kallay' self grafts failed to produce new tracheal
cells during the first 30 days after grafting. A similar but shorter
delay was also found in 'Fountain' self grafts. In 'Hetzii' self
grafts, a plateau in cell production between 50 and 60 days in normal
wood, and between 40 and 50 days in wound induced wood, was observed.

In most instances, scion cell counts of all clones increased
significantly with increasing ploidy level of the understock; the
reverse occurred when 'Pfitzeriana Kallay' was the scion.

In most graft unions 'Hetzii' scions and understocks produced
greatest cell numbers. In wound tissue, however, 'Pfitzeriana
Kallay' understocks produced greatest numbers of cells.

In 'Fountain' on 'Pfitzeriana Kallay' grafts, cell numbers were
significantly higher than those of either self graft; this thought to
be an expression of the effect observed when 'Fountain' extract was
applied to 'Pfitzeriana Kallay' self grafts.

Ruthenium red Staining revealed that pectic substances were con—
centrated in the middle lamella of mature tracheids but very diffuse
in undifferentiated tissue.

In addition to adding to our basic knowledge of the cytology,
histology, and sequence of events occurring during graft healing of
certain clones of Juniperus L., new procedures showing promise for
future studies were tested, and certain new avenues of approach in

graft studies were brought to light.

 

w--..—.._

 

APPENDIX B

Appendix Table B4.

Analyses of variance for stock—scion mineral

content ratios of juniper grafts sampled at
regular intervals after grafting*

 

 

 

 

 

5%
SOURCE D.F. S.S. M.S. F.
Total 269 6,034.86
Dates 9 118.13 13.13 .75 N.S.
Combinations 8 1,066.51 133.31 7.61**
D X C 72 1,695.01 23.54 1.34 N.S.
Error 180 3,155.21 17.53

Potassium
SOURCE D.F. S.S. M.S. F.
Total 269 5,979.06
Dates 9 172.43 19.16 .919 N.S.
Combinations 8 637.22 79.65 3.820**
D X C 72 1,416.30 19.67 .940 N.S.
Error 180 3,753.11 20.85

Magnesium
SOURCE D.F. S.S. M.S. F.
Total 269 8,017.15
Dates 9 510.93 56.77 3.21**
Combinations 8 2,096.13 262.02 14.80**
D X C 72 2,222.25 30.87 1.74**
Error 180 3,187.84 17.71

 

 

* . . .
AnalySis of variance based upon arCSin transformed data.