AN ABSTRACT OF THE THESIS OF MYCQRRHIZAE. …

AN ABSTRACT OF THE THESIS OF

Sandra Gonzalez-Caldwell for the degree of Master of Science in

Horticulture presented on December 28. 1989.

Title: ROOTING OF PEAR HARDWOOD CUTTINGS USING VESICULAR-ARBUSCULAR

MYCQRRHIZAE. AGROBACTERIUM RHIZOGENES AND ROOTING HORMONES.

Abstract approved: _ . ^ Dr. Porter Lombard

Rooting experiments were conducted with hardwood cuttings of Old

Home x Farmingdale (OHXF) pear rootstock selections 217 and 282 to

determine the effects of rooting hormones, VA mycorrhizal (VAM) fungi

and the bacteria Agrobacterinm rhizopenes applied to the cuttings or

added to the rooting medium.

Mycorrhizal fungus inoculum was generated in pot cultures of pear

roots collected from pear trees at two locations. Pot cultures

contained spores of Glomus fasciculatum. G. microaggregatum. G.

intraradices. G. caledonium. G. mosseae. G. occultum. Acaulospora

trappei. and a Glomus species similar to G^. pallidum: the first three

species were most prevalent.

Inoculation with VA mycorrhizal (VAM) fungus inoculum from pear

roots did not increase, and sometimes reduced rooting of pear

cuttings. This negative influence appeared to be the effects of other

microbial components of the pot culture contents, since few

mycorrhizae formed on cutting roots.

Of three strains of Aprobacterium rhizogenes tested, strain TR105

improved rooting most, compared to untreated cuttings. Rooting

enhancement, however, was only sometimes as good as but never better

than that achieved with rooting hormones.

OHXF selection 282 always rooted better than selection 217, but

both rooted best with the addition of rooting hormones, regardless of

the rate or source. Rooting response was quantified by nine

parameters including root number, root length, percent of root

branching, survival, a rating of rooting based on a scale of 1 to 4,

root dry weight, root area, shoot number, and shoot dry weight.

Hormone treatments alone gave 71-96 % survival of cuttings compared to

8-21 % without hormones.

Some effects were detected between hormone treatments with A.

rhizogenes and/or VAM fungus inoculum, such as increased rooting of

cuttings with A^. rhizogenes when no hormone was applied, and a

decrease when VAM were used in combination with the powder.

These studies demonstrate that OHXF pear cuttings can be

successfully rooted with hormone treatments, but the microbes A.

rhizogenes and VAM fungi need further experimentation to demonstrate

any possible value. Adding inoculum after rooting from hormone

treatments could maybe enhance subsequent growth of roots and shoots.

ROOTING OF PEAR HARDWOOD CUTTINGS USING

VA MYCORRHIZAL FUNGI. AGROBACTERIUM

RHIZOGENES AND ROOTING HORMONES

by

Sandra Gonzalez-Caldwell

A THESIS

submitted to

Oregon State University

In partial fulfillment of

the requirements for the

degree of

Master of Science

Completed December 28, 1989

Commencement June 1990

APPROVED:

i.f ■ ' -"tf^ ■—i, - t—

Professor of Horticulture in charge of major

T^T1 a*' Head of Denaxtment of Horticulture

Dean of Gc^ctiate School 'H^^V,

Date thesis is presented December 28.1989

Typed by Sandra Gonzalez-Caldwell

Acknowledgements

I wish to express thanks to my major professor, Dr. Porter

Lombard, for advice and help during this research project.

Deep gratitude goes to Dr. Robert Linderman, my academic co-advisor,

in whose laboratory and greenhouse facilities this research was

accomplished, for moral support, advice and guidance throughout, as

well as technicians, researchers and graduate students from the

Horticultural Crops Research Laboratory who aided me in so many

different ways, enriching my learning experience and making it easier

for me to work in new and unfamiliar areas.

I wish to also thank Dr. James Trappe for help with VAM fungus

identification, and Dr. Larry Moore and his lab for supplying the

Agrobacterium rhizogenes strains and training necessary to work with

these bacteria.

I am indebted to good friends in the Horticulture Department, Dr.

Robert Stebbins and his wife who, thanks to their kindness and help,

have made possible the completion of this degree. In all, my studies

in the United States have been a wonderful opportunity for growth,

both academic and personal aspects.

TABLE OF CONTENTS

I. INTRODUCTION 1

II. MATERIALS AND METHODS 6

Preparation of the VAM inoculum 6

Identification of VA mycorrhizae from pear roots 7

Source and method of application of A. rhizogenes 8

Hormone formulations and concentrations 9

Plant material 9

Rooting media, containers and greenhouse conditions .... 10

Statistical design, treatments 10

Evaluation 11

a. Root production 11

b. VAM colonization 13

c. Retrieval of A. rhizogenes 13

d. Leaf and shoot growth 13

e. Survival 13

III. RESULTS AND DISCUSSION 15

Effect of rootstock cultivar 15

Detection of VAM in rooted cuttings 16

Effect of VAM on rooting 16

Retrieval of A. rhizogenes 17

Effect of A. rhizogenes on rooting 18

Effect of hormones on rooting 18

IV. BIBLIOGRAPHY 37

V. APPENDIX 41

LIST OF FIGURES

Figure No. Page

1. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root number of mist propagated hardwood cuttings of OHXF 217 19

2. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root number of mist propagated hardwood cuttings of OHXF 282 20

3. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root length of mist propagated hardwood cuttings of OHXF 217 21

4. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root length of mist propagated hardwood cuttings of OHXF 282 22

5. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on % root branching of mist propagated hardwood cuttings of OHXF 217 23

6. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on % root branching of mist propagated hardwood cuttings of OHXF 282 24

7. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on survival index of mist propagated hardwood cuttings of OHXF 217 25

8. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on survival index of mist propagated hardwood cuttings of OHXF 282 26

9. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on rooting rating of mist propagated hardwood cuttings of OHXF 217 27

10. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on rooting rating of mist propagated hardwood cuttings of OHXF 282 28

11. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root dry weight of mist propagated hardwood cuttings of OHXF 217 29

Figure No. Page

12. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root dry weight of mist propagated hardwood cuttings of OHXF 282 30

13. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root area of mist propagated hardwood cuttings of OHXF 217 31

14. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root area of mist propagated hardwood cuttings of OHXF 282 32

15. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on shoot number of mist propagated hardwood cuttings of OHXF 217 33

16. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on shoot number of mist propagated hardwood cuttings of OHXF 282 34

17. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on shoot dry weight of mist propagated hardwood cuttings of OHXF 217 35

18. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on shoot dry weight of mist propagated hardwood cuttings of OHXF 282 36

LIST OF APPENDIX TABLES

Table No. Page

1. Effect of VA mycorrhizae, Agrobacterium rhizoeenes. and rooting compounds on root numbers, root length, and % of root branching of mist propagated hardwood cuttings of OHXF 217 41

2. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root numbers, root length, and % of root branching of mist propagated hardwood cuttings of OHXF 282 42

3. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on survival index, rooting rating, and root dry weight of mist propagated hardwood cuttings of OHXF 217 43

4. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on survival index, rooting rating, and root dry weight of mist propagated hardwood cuttings of OHXF 282 44

5. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root area, shoot number, and shoot dry weight of mist propagated hardwood cuttings of OHXF 217 45

6. Effect of VA mycorrhizae, Agrobacterium rhizogenes. and rooting compounds on root area, shoot number, and shoot dry weight of mist propagated hardwood cuttings of OHXF 282 46

ROOTING OF PEAR HARDWOOD CUTTINGS USING VESICULAR-ARBUSCULAR MYCORRHIZAE,

AGROBACTERIUM RHIZOGENES AND ROOTING HORMONES

I. INTRODUCTION

The use of clonal rootstocks, in contrast to seedlings, is of great

value in tree fruit production. Those recommended for each species and

cultivar have been selected for their characteristics such as disease

resistance, performance under unfavorable soil and climatic conditions,

scion compatibility and tree size-regulating capacity. Vegetative

propagation of these clonal materials leads to orchard uniformity, which

greatly contributes to cost-effective production. In cultivars where

layering is not economical, rooting of stem cuttings is the next best

alternative. Use of hardwood cuttings should be an efficient, relatively

simple and inexpensive method of clonal propagation, requiring minimal

investment and substantially less specialized labor compared to other

promising alternatives such as tissue culture, especially when dealing with

limited quantities which are frequently required with perennial tree crops.

Orchards can be economically productive for over 15 years.

Plant materials, even within the Rosaceae family (deciduous fruits),

differ widely in their propensity to root from stem cuttings. Frequently,

rootability is markedly influenced by the mother plant and weather

conditions (Hansen,1987), harvest date, dilution and nature of applied

hormone and rooting bed conditions (Howard, 1968; Hartmann and Kester,

2

1985). Hardwood cuttings of pear are difficult to root because of such

factors.

Commercial rooting of pear rootstock cuttings has involved the use of

lH-indole-3-butanoic acid (IBA) dips on hardwood (Westwood and Brooks,

1963) . Two methods used in their propagation trials of Old Home X

Farmingdale (OHXF) hardwood cuttings were: a) cuttings harvested in the fall

after chilling commenced, prior to full chilling using 100 to 200 ppm IBA in

a 24 h soak; and, b) dormant cuttings pruned in late January, after chilling

requirements were met, treated with 1000 to 2000 ppm IBA in 5 sec dips.

Rooting success in their trials ranged from 10 to .78 %, varying with the

size and condition of the plant material. To be acceptable in commercial

rooting of cuttings, they suggest a minimum of 50 % success. The OHXF

rootstock series has been pointed out as displaying a wide spectre of

propensity to root from stem cuttings, from 0 to 80 % (Westwood, pers.

comm.). There is need to develop treatments which could improve the

adventitious rooting of these difficult-to-root materials, and in the

process, possibly improve the understanding of physiological events

involved. With these purposes in mind, vesicular-arbuscular mycorrhizae

(VAM) and the bacterium Agrobacterium rhizopenes were selected for study.

Mycorrhizal fungi have been found to produce growth-regulating

substances and other organic compounds in the rhizosphere (Cooper,1985;

Ek,1983; Rouillon,1985; Madej,1985) and thus were studied as potential

rooting aids for many types of cuttings (Linderman and Call,1977; Barrows

and Roncadori,1977; Branzanti et al,1985; Cooper,1985; Powell and

Santanakrishnan,1986; Verkade,1986).

3

A. rhizopenes. formerly regarded as a pathogen, displays extraordinary

ability to transfer certain parts of its DNA to the host genome. This

transfer (T) DNA has been shown to confer auxin responsiveness to the

transformed plant tissues enabling root differentiation in the presence of

auxin, an event that did not occurr in untransformed material (Cardarelli et

al., 1987).

Vesicular-arbuscular mycorrhizal (VAM) fungi, so-called because of the

structures formed within root cortical tissues (arbuscules and vesicles) are

fungal symbionts in the roots of many plants. Vesicles are storage organs

formed on internal hyphae while arbuscules are complex haustoria-like

structures involved in nutrient exchange within the plant cell. VA

mycorrhizae differ from ectomycorrhizae in that the fungal symbionts are

obligate, penetrate plant cells, and do not significantly alter root

morphology. They achieve long term survival in soil by producing thick-

walled chlamydospores.

Aprobacterium rhizogenes. first studied as the causal agent of 'hairy

root' disease (Suit, 1933) in dicots (DeCleene,1981), is a bacterium which

causes great proliferation of roots in wound sites, by means of a plasmid-

mediated genetic transformation (Moore, 1979). Aside from the impressive

potential that the Ri plasmid and its peculiarities represent for molecular

biology (Birot, 1987; Klee, 1987; Zambryski, 1989), the capability of

eliciting prolific rooting in host plants could prove to be a useful tool in

plant propagation. This has been attempted with success (Diana,1986;

Strobel,1985,1987; Bassil and Proebsting, pers. communic), thus harnessing

the very quality (Ark, 1961) that caused it to be considered pathogenic.

Tepfer's (1984) finding that A^ rhizogenes- transformed plants displayed

4

shortened internodes (dwarfing) merits verification for fruit trees;

rootstocks displaying such traits are rare and desirable.

A number of works confirm the dynamic interactions occurring between

rhizosphere microorganisms and mycorrhizae (Linderman, 1988; and Linderman

and Paulitz, a review in press). In many cases, rhizobacteria and

mycorrhizae were shown to exert a mutually positive influence on each other,

either directly by increasing availability of certain nutrients or growth

substances (Barea,1975; Raj,1981; Leyval,1988) or indirectly by suppressing

potentially deleterious organisms (Cook and Baker,1983). Whenever such

situations arise, it is valid to assume that the host plant would also

benefit, whether from the absence or decreased populations of certain

pathogens and/or the added effect/s that mycorrhizae and associated bacteria

may exert, either directly or through mutual complementation.

In the case of a stem cutting, the potential for adding beneficial

microorganisms is even more significant, since in the absence of roots it is

severely stressed. Cutting survival depends on rapid morphological changes

in response to small variations in many factors, the most important being

phytohormones (Hartmann and Kester, 1985). Rhizospheric microorganisms

which could consistently stabilize host response (and interact

mutualistically) leading to enhancement of adventitious rooting processes

could be extremely valuable, not only for improving the percentage and

quality of rooting but also for safeguarding against potential stress

problems, many of which cannot be detected until too late (e.g. pathogenic

infection of plant tissue, drought, nutrient stress, etc.). Caesar and Burr

(1987) showed that various bacterial strains promoted growth of clonal

rootstocks of apple. Many species of higher plants have been shown to form

metabolites called phytoalexins when exposed to fungi or mechanical wounding

(Keen,1981). Certain phytoalexins present in concentrations below their

antibiotic activity have been shown to act synergistically with lH-indole-3-

acetic acid (IAA) to promote adventitious rooting (rooting cofactor

activity) in mung bean bioassays, (Yoshikawa et al.,1986). This fact might

partially account for the positive effects of certain bacteria and fungi on

plant rooting processes.

The purpose of this study was to answer the following questions:

a. Is rooting of Old Home x Farmingdale pear hardwood cuttings influenced by

VAM?

b. Does Aprobacterium rhizopenes affect rooting?

c. How do different concentrations and formulations of plant growth

regulators compare to the above treatments?

d. Do any of the above factors interact in any way in adventitious rooting?

II. MATERIALS AND METHODS

An experiment to study the rooting of hardwood cuttings of the pear

rootstock series Old Home x Farmingdale (Westwood, 1982), developed in

Oregon and unique for its resistance to the diseases pear decline and fire

blight, was begun in the greenhouse in November 1988. The treatments

included VAM fungi collected from pear orchards, three strains of

Aprobacterium rhizopenes and three phytohormone treatments.

Preparation of the VAM inoculum:

Pear roots were removed from trees in several orchards during fall and

winter 1987/1988, washed in water, cleared and stained for detection of VAM

colonization (Phillips and Hayman, 1970). Colonized roots were surface-

sterilized using 0.5 % sodium hypochlorite and cut into 1 cm pieces to be

used as inoculum to multiply the symbionts in plant pot cultures. The

latter were started in the following way: half-gallon pots filled with

pasteurized (air-steamed 1 h at 60 C) sand in which three cavities, 2.5 cm

wide x 10 cm deep, were made. These cavities were 75 % filled with VAM root

inoculum, topped with sand. One seed of subterranean clover (Trifolium

repens L.), previously surface-sterilized 10 min. with .5 % sodium

hypochlorite solution, was placed on each inoculum core. Cultures were

maintained under greenhouse conditions for 3.5 months and watered and

fertilized weekly with Long Ashton Plant Nutrient Solution (P content

modified to 11 ppm). The pots were allowed to dry and were then harvested.

Aerial plant parts were discarded, the roots were cleared and stained to

determine the degree of VAM colonization. The entire volume of sand and

roots divided into 1 cm pieces was thoroughly mixed and stored under dry,

7

cold conditions (4 C) for later use. VAM - free medium for controls was

made by washing sand from pot cultures with water, sieving the resulting

slurry through a 38 micron metal sieve and blending the sieved liquid with

pasteurized sand.

Identification of VA mvcorrhizae from pear roots:

The pot cultures of VAM that served as inoculum for the experiment were

made from surface-sterilized pear roots gathered from orchards in Medford

and in different locations at the National Germplasm Repository [USDA-ARS],

Corvallis, OR. Dr. James Trappe, from the Department of Forest Science,

Oregon State University, kindly indentified the VAM in those cultures based

on examination of spores contained therein. The findings were as follows:

Sample(Location) VAM species

1. (Medford) Glomus fasciculatum (Thaxter) Gerd. &

Trappe emend. Walker & Koske.

2. ( " ) a.G. microapprepatum Koske, Gemma & Alexis.

b.G. ca'ledonium (Nicol. & Gerd.) Trappe &

Gerd.

3. ( " ) G. fasciculatum.

4. ( " ) G. fasiculatum ? (if so, spores very

young).

G. fasciculatum.

None.

G. intraradices Smith & Schenck.

G. fasciculatum.

5. ( tl )

6. ( 11 )

7. ( 11 )

8. ( II )

Sample(Location)

9.

10.

11.

12.

13.

18.

19.

20.

21.

22.

23.

24.

" )

" )

Corvallis)

14. ( ' It )

15. ( It )

16. It )

17. 11 )

VAM species

G. microagpregtatum.

Too few spores to identify.

Spores found mainly in roots, probably G.

intraradices.

Acaulospora trappei Ames & Linderman.

a.A. trappei.

b.Glomus sp. (too few spores to identify)

G. cf pallidum Hall or poss. undescribed.

G. mosseae. plus possibly one or two other

immature or otherwise unidentifiable spp.

a.G. fasciculatum.

b.G. mosseae.

G. occultum Walker.

G. fasciculatum.

G. intraradices.

G. fasciculatum.

Source and method of application of A. rhizopenes:

Strains A 4, A 4783 and TR 105 were provided by Dr. Larry Moore,

Department of Botany and Plant Pathology, Oregon State University. Strain A

4 was originally isolated by Dr. P. Ark (Univ. of California, Berkeley) from

9

roses, A 4783 was discovered by M. Canfield (Oregon State Univ.) from

carrot, and TR 105 was of unknown origin. Petri dishes with mannitol

glutamate medium (Keane et al., 1970) were inoculated with the bacteria and

incubated for 72 h at 25 C. A cell suspension of each strain was made by

pouring sterile water on these dishes and rubbing with a glass rod. The

resulting cell suspension was poured into tubes and vortexed until

homogenized. Sterile water was added to obtain the desired reading of 70 on

a Klett colorimeter (approx. 3 x 10 colony forming units/mL).

Approximately 30 plates were used to prepare 2.25 L of bacterial suspension

for each strain. The suspensions for inoculation were prepared on the

daythey were used; 5 mL of each was pipetted into the center of each pot

shortly before cuttings were 'stuck'.

Hormone formulations and concentrations:

Indole butyric acid (IBA), dissolved in 50% ethanol, was used at 5000

and 2500 ppm (Westwood and Brooks, 1963) as was a dry hormone preparation,

referred to as Hormodust, containing 1000 ppm each of IBA and NAA in talc.

Cuttings were dipped to a depth of 0.5 cm for 5 sec in the solutions or

powder, (Howard and Nahlawi, 1970), excess liquid or powder was flicked off

immediately, and the cutting was then stuck.

Plant material:

Cuttings of the pear rootstock series Old Home x Farmingdale (OHXF),

genotype selections 217 and 282, provided by Patchwork Nursery, Forest

Grove, Oregon, were harvested on October 22, 1988 and hand defoliated as

necessary (Westwood and Brooks, 1963). The topmost and lowest 10 cms from

each cutting were removed. Stems with calipers less than 0.7 cm and those

greater than 1.0 cm were discarded (Marini, 1983). The bottom cut was made

10

perpendicular to the stem, 0.5 cm below the first leaf node, resulting in a

final length of 15 cm. All cuts were made just before sticking.

Rooting media, containers and greenhouse conditions:

The rooting medium consisted of 4 parts perlite, 3 parts Douglas fir

sawdust and 1 part steam sterilized Hypnum peat (Biermann and

Linderman,1983) blended in a twin-shell blender and adjusted to pH 7 with

CaC03 (Davis et al.,1983). Black plastic 'band' pots measuring 6 x 6 x 13

cm were placed in plastic flats on a greenhouse bench equipped with bottom

heat cables (24 C) and intermittent mist. The bench was covered entirely

with a plastic tent rising 120 cm above the cuttings. Air temperature

during the first three months was maintained at 10 C, but thereafter the

temperature was allowed to rise during daylight hours.

Bands were filled with rooting media, leaving a central cavity of 7 cm

deep x 2 cm wide, where 18 mL of VAM inoculum from the pear root sources or

non-mycorrhizal medium were added. Cuttings were stuck so that their lower

ends were in the inoculum.

Statistical design, treatments:

The design was a factorial in a randomized block for each pear genotype

with 32 treatments of 24 cuttings per treatment, totalling 1536 units. All

cuttings were placed on the same greenhouse bench, randomly arranged in 4

replicates of six cuttings each. The treatments were applied to each

rootstock cultivar, as follows:

11

Treatment VAM HORMONE A. rhizopenes

1. 2. - - A 4 3. - - A 4783 4. - - TR 105 5. - IBA 2500 - 6. - IBA 2500 A 4 7. - IBA 2500 A 4783 8.. - IBA 2500 TR 105 9. - IBA 5000 - 10 - IBA 5000 A 4 11. - IBA 5000 A 4783 12. - IBA 5000 TR 105 13. - 'HORMDST' - 14. - 'HORMDST' A 4 15. - 'HORMDST' A 4783 16. - •HORMDST' TR 105 17. + - - 18. + - A 4 19. + - A 4783 20. + - TR 105 21. + IBA 2500 - 22. + IBA 2500 A 4 23. + IBA 2500 A 4783 24. + IBA 2500 TR 105 25. + IBA 5000 - 26. + IBA 5000 A 4 27. + IBA 5000 A 4783 28. + IBA 5000 TR 105 29. + 'HORMDST' - 30. + 'HORMDST' A4 31. + 'HORMDST' A 4783 32. + 'HORMDST' TR 105

Evaluation:

The following observations were made:

a. Root production - Root number, average root length, root area, rooting

rating and presence of lateral roots were quantified. Each cutting was

extracted individually from the rooting medium and carefully washed in cold

water. The roots were severed from the cutting and placed on a sheet of

clear celulloid under a white background and photocopied (Collins et al.,

1987). All separate roots were placed in the same direction. This provided

12

a permanent record of the configuration, number, average length and lateral

branching of roots. The root images, as photocopies, were later analyzed

with a Delta-T brand area meter (Harris and Campbell, 1989) and the

measurement obtained is hence referred to as root area. The area meter was

calibrated to read with precision to 0.1 cm on a cm2 scale.

An artificial scale to rate rooting response, ranging from 1 to 4 was

used as follows:

1= base of cutting unchanged, no rooting.

2= presence of callus only, no rooting.

3= rooted, root lengths less than 5 cm

4= rooted, root lengths over 5 cm

Lateral branching of roots was estimated as a means of detecting'hairy

root'-like proliferation, a feasible occurrence in roots treated with A.

rhizoeenes. It involved a subjective evaluation of the profusion,

ramification and size of lateral roots emerging from roots originating at

the cutting base, expressed in percentage (100 % corresponding to the

maximum and 0% to the minimum). In order to standarize readings the root

photocopies were used instead of the actual roots, thereby confining the

observations to one physical plane.

Total fresh weight of roots from each cutting was then measured.

However, representative samples for clearing and staining for VAM

colonization were removed after that step, and the remainder oven dried at

70 C for 72 hours. Sample fresh/dry weight ratios were used to calculate

total dry weight. This conversion was used to add back the weight of the

roots removed for VAM colonization analyses.

13

b. VAM colonization - VAM colonization was evaluated microscopically after

clearing and staining with Trypan blue in lactoglycerin (Phillips and

Hayman,1970). The percent root length with VAM was determined by the method

of Biermann and Linderman (1981).

c. Retrieval of A. rhizogenes - Various techniques were used to retrieve A,

rhizopenes from roots and/or rooting medium rhizosphere samplings. Another

approach used was with water-washed root macerates, surface-sterilized

(using 0.5 % sodium hypochlorite solution) or not, prepared either with

mortar and pestle or by applying the hand-held 'tissue disrupter' (Agdia

Corp.; Elkhart, Indiana 46154) over roots placed for that purpose in plastic

self-locking bags into which 10 cc of sterile water was initially placed.

The supernatants of each of the resulting samples, both rooting media or

root macerations, were made into acqueous supensions diluted to 10-1, 10-2

and 10-3, and plated on Petri dishes containing two different agar selective

media (Brisbane and Kerr, 1983), one containing malachite green (selective

for Biovar II), the other gentian violet (Biovar I specific).

d. Leaf and shoot growth:

Numbers of shoots per cutting were recorded, and the total lengths

recorded for each cutting. Freshly severed leaves and shoots laid flat

under a clear glass sheet were measured with the Delta-T area meter,

calibrated to read with precision of 0.1 cm on a cm2 scale. Shoot and leaf

fresh weight was recorded, then dried for 72 h at 70 C for dry weight

determination.

e. Survival:

Survival was determined by counting, within each replicate, those

cuttings that have achieved sufficient root development to ensure survival.

14

This rating was subjective in that it did not consider cuttings which had

only callused or had very sparse rooting; it aimed to evaluate survival

capacity if transplanted to a larger container or into a field situation

15

III. RESULTS AND DISCUSSION

All the above parameters were evaluated, but shoot length was

disregarded because variation was too great for it to be considered a

reliable indicator of treatment effects. The values for shoot area

indicated a positive trend in treatments which included rooting compounds,

but the variability within treatments precluded them being statistically

significant.

A parameter resulting from the artifact of multiplying average root

length x root number for each cutting was tested as an additional estimation

of root development, but was later disregarded because it showed the same

patterns as root length measurement.

Effect of rootstock cultivar

Cuttings of OHXF 282 had higher survival and rooting ratings than OHXF

217, after 3.5 months in the rooting chamber (Figures 7, 8; and 9, 10

respectively, and Appendix Tables 3 and 4). The parameter "survival" refers

to the 'take' of each cutting, while rooting rating, though related, is more

descriptive of morphological changes which occurred (or not) at the base,

(e.g. callusing; formation of short, stubby roots; or the long, healthy ones

essential for good quality nursery stock). The survival index means for

OHXF 217 and 282 were .62 and .71, respectively, giving a clear indication

that the 'take' of OHXF 282 is inherently the better of the two. The rooting

rating means also reflect that tendency, at 2.99 and 3.27 for 217 and 282,

respectively.

Root number averages for each rootstock were 3.86 and 5.82; root length,

5.66 and 6.27; root branching, 13.06 and 18.79, the higher of each pair of

16

values invariably corresponding to OHXF 282. These data confirm what the

nurseryman who provided the cutting material communicated, regarding the

better rooting qualities of OHXF 282 over 217. Westwood and Brookes (pers.

communic.) also indicated a wide range of adventitious rooting ability

within this line of rootstocks.

Detection of VAM in rooted cuttings:

Roots of OHXF 217 cuttings yielded no detectable VAM colonizations,

while those of OHXF 282 showed root length colonizations ranging between 5

and 10 per cent in four root samples, corresponding to treatments 19, 20, 21

and 28. It is assumed that the levels of colonization would have been

higher should the cuttings have been evaluated at a later date, but such

practices are not economically feasible for commercial nurseries. The low

rates of colonization found made it impossible to identify the species of

VAM involved.

In this study, inoculation with VAM fungi had no effect in most cases.

Since the inoculum was generated in pot culture from colonized pear roots,

we assume the fungi would be compatible with roots of pear cuttings.

However, since practically no mycorrhizae were detected in cutting roots,

other factors could have precluded their formation. Possible explantions

for lack of colonization could be factors affecting inoculum potential

including medium pH, spore dormancy, physical placement in relation to

roots, delayed formation of roots, etc.

Effect of VAM on rootinp:

Controls without VAM fungus inoculation actually rooted better than

those containing VAM inoculum for the parameters survival and rooting rating

(Figures 7, 8, and 9, 10, respectively; Appendix Tables 3 and 4). Values

17

for root number followed the same trend, with overall means for VAM

treatments being 4.45 compared to 5.26 for controls.

VAM fungus treatments which included the powdered hormone formulation

showed lower responses for rooting rating, shoot dry weight, root length,

root number and root branching at the P-0.001 level of significance. For

instance, means for root number were 6.33 for no VAM plus powder formulation

versus 3.75 for VAM with powder (Figures 1 and 2). In contrast, results for

IBA at 5000 ppm were 7.26 and 6.66, respectively, without and with VAM

inoculum.

Rooting rating results reflect the same tendency, as shown by the means:

3.41 (no VAM inoculum plus powder) versus 2.88 (VAM inoculum plus powder).

There was little difference between the rooting rating for the other

hormones with or without VAM fungus inoculum. An explanation could be that

the VAM inoculum exerted some negative effect on the hormone contained in

the powder formulation, either through a degrading or immobilizing action.

The fact that this is the only formulation studied which contains NAA

invites speculation as to the possible effect on the rhizosphere flora or

the microbes contained in the VAM pot culture inoculum.

A second relationship was found between VAM inoculum and Agrobacterium

which indicated that rooting rating was decreased with VAM added to the

rooting medium in the absence of bacteria or with strain TR 105 (Figures 9

and 10; Appendix Tables 3 and 4). It could be speculated that, since no

other root parameters are affected, the factor being influenced is the

formation of callus, which is only evaluated by rooting rating.

Retrieval of A. rhizogenes:

18

All attempts to recover and identify Aprobacterium strains failed due to

the extensive proliferation of great numbers of bacterial colonies after 72

h incubation at 26 C, most of which did not have Agrobacterium

characteristics. Controls responded in the same way as did treatments. A

strain marked for antibiotic resistance might be considered for future

studies.

Effect of A. rhizopenes on rooting:

The inclusion of any strain of A^. rhizopenes in the rooting medium

increased survival, rooting rating and root dry weight, (Figures 7, 8, 9,

10, 11 and 12; Appendix Tables 3 and 4). The most effective strains were A

4783 and TR 105. The survival rate of cuttings with any bacterial inoculum

ranged from 65 to 77 per cent; average survival on cuttings without

bacterial inoculum was 58 %. The positive effect of the bacterial

suspension could be due to enhanced initiation of adventitious roots due to

transformation of cells within the cutting. Confirmation of transformation

by opine analysis was not done, however.

An relationship between Agrobacterium and rooting compounds was found

for the parameters root number, root length, root branching, rooting rating,

shoot number and shoot dry weight. When Agrobacterium was applied to the

rooting medium without hormones, there was a slight increase in root

initiation and development.

Effect of hormones on rooting:

Use of IBA at 2500 or 5000 ppm or the dry hormone formulation increased

the rooting of both cultivars of pear cuttings regardless of the parameters

measured (P=0.001). In contrast, none of the biological agents were as

effective.

OLD HOME X FARMINGDALE 217: ROOT NUMBER 19

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OLD HOME X FARMINGDALE 217: ROOT LENGTH 21

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Fiq. 3. Effect of VA mycorrhizae, Aqrobacterium rhizoqenes and rooting compounds on the root length of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.

OLD HOME X FARMINGDALE 282: ROOT LENGTH ??

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OLD HOME X FARMINGDALE 217: ROOT BRANCHING

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OLD HOME X FARMINGDALE 282: ROOT BRANCHING 24

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OLD HOME X FARMINGDALE 217: SURVIVAL

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Fig. 7. Effect of VA mycorrhizae, Aarobacterium rhizogenes and rooting compounds on survival index of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.

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27 OLD HOME X FARMINGDALE 217; ROOTING RATING

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OLD HOME X FARMINGDALE 282: ROOTING RATING 28

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Fig. 10. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on rooting rating of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.

29

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OLD HOME X FARMINGDALE 282: ROOT DRY WEIGHT

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31 OLD HOME X FARMINGDALE 217: ROOT AREA

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Fig. 13. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on root area of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.

OLD HOME X FARMINGDALE 282: ROOT AREA

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ESaNO BACT .CZIA4 EaA4783

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^Q ab /

>>


VAM

25.00

20.00

15.00

10.00

0.00

25.00

20.00

15.00

10.00

5.00

0.00

Fig. 14. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on root area of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.

OLD HOME X FARMINGDALE 217: SHOOT NUMBER

A) 1.80

1.60.

■i->

z. 1.20 o

^ 1-00

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B)

: 0.40 > : 0.20

0.00

ESS NO BACT IZDA4 Â4783

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/ ab '

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NO VAM


VAM

1.80

1.60

1.40

1.20

1.00

0.80

0.60

0.40

0.20

0.00

Fig. 15. Effect of VA mycorrhizae, Aqrobacterium rhizoqenes and rooting compounds on shoot number of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.

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B)

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Fig. 17. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on shoot dry weight of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.

OLD HOME X FARMINGDALE 282: SHOOT DRY WEIGHT

A) 1.20

1.00

DO

w 0.80

£ 0.60

| 0.40 in

% 0.20

0.00

1.20

B) 1.00 t—*

4J 0.80 3

■o 0.60

o i 0.40

0) >0.20

0.00

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Â4783 BETR105

I

hi hi

NO H0RM IBA 2500 IBA 5000 POWDER

NO VAM •

ES NO BACT IZHA4 Â4783 OMTRIOS

ad

i

1 /d9


VAM

1.20

1.00

0.80

0.60

0.40

0.20

0.00

1.20

1.00

0.80

0.60

0.40

0.20

0.00

Fig. 18. Effect of VA mycorrhizae, Agrobacterium rhizogenes and rooting compounds on shoot dry weight of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each bar represents the mean of 24. Columns with the same letter/s are not significantly different at P=0.05 using mean standard error.

37

IV. BIBLIOGRAPHY

Ark, P.A. and Thompson,J.P. (1961). Detection of hairy root pathogen, Agrobacterium rhizogenes. by the use of fleshy roots. Phytopathology, 51:69-71.

Barea.J.M.; Azcon.R. and Hayman.D. (1975). Possible synergistic interactions between Endogone and phosphate-solubilizing bacteria in low-phosphate soil. In Sanders,F.E.; Mosse.B. and Tinker,P.B.(eds), Endomycorrhizas. Academic Press, London, pp.409- 417.

Barrows,J.B. and Roncadori.R.W. (1977). Endomycorrhizal synthesis by Gipaspora margarita in poinsettia. Hycologia 69 (6): 1173- 1184.

Biermann.B. and Linderman,R.G. (1983). Effect of container plant growth medium and fertilizer phosphorus on establishment and host growth response to vesicular-arbuscular mycorrhizae. J. Am. Soc. Hortic. Sci. 108 (6): 962-971.

Biermmann.B. and Linderman, R.G. (1981). Quantifying vesicular- arbuscular mycorrhizae: a proposed method towards standarization. New Phytol. 87, 63-67.

Birot.A.M.; Bouchez.D.; Casse-Delbart.F.; Durand-Tardif,M.; Jouanin.L.; Patout.V.; Robaglia.C.; Tepfer, D.; Tepfer.M.; Toumeur.J. and Vilaine.F. (1987). Studies and uses of the Ri plasmids of Agrobacterium rhizogenes. Plant Physiol. Biochem. 25: 323-335.

Branzanti.B.; Cristoferi.G.; Zocca.A. and Zambonelli.A. (1985). Ectomycorrhizal fungi and IBA effects on fruit rootstock rooting. In Proc. VI N. Am. Conf. on Mycorrhizae, p. 324. June 25-29, 1984, Bend, OR. 471pp. Brisbane,P.G. and Kerr, A. (1983). Selective media for three biovars of Agrobacterium. J. Appl. Bacteriol. 54:425-431.

Caesar,A.J. and Burr.T.J. (1987). Growth promotion of apple seedlings and rootstocks by specific strains of bacteria. Phytopathology 77: 1583-1588.

Collins,R.P.; Gregory,P.J.; Rowse.H.R.; Morgan,A. and Lancashire,B. (1987). Improved methods of estimating root length using a photocopier, a light box and a bar code reader. Plant Soil 103, 227-280.

Cook.R.J. and Baker, K.F. (1983). The nature and practice of biological control of plant pathogens. The American Phytopathological Soc. Press, St. Paul, MN. 539 pp.

38

Cook.R.J. and Baker, K.F. (1983). The nature and practice of biological control of plant pathogens. The American Phytopathological Soc. Press, St. Paul, MN. 539 pp.

Cooper,K.M. (1985). Physiology of VAM associations. IV. Hormonal effects. In "VA Mycorrhizae', Eds. Powell,C.L. and Bagyaraj,D.J. CRC Press, pp. 173-186.

Davis,E.A.; Young, J.L. and Linderman.R.G. (1983). Soil lime level (pH) and VA-mycorrhiza effects on growth responses of sweetgum seedlings. Soil Sci. Soc. Am. J. 47:251-256.

De Cleene.M. and De Ley.J. (1981). The host range of infectious hairy-root. The Botanical Review 47: 147-194.

Diana,G. (1986). [The transmission of Aprobacterium rhizogenes plasmids in propagation of olive cuttings.] Trasmissione di plasmidi di Aprobacterium rhizogenes nella propagazione di talee di olivo. Annali dell'Istituto Sperimentale per 1'Olivicultura (1981/1983) 7, 1-11.

Ek,M.; Ljunquist.P.O. and Stenstrom.E. (1983). Indole-3-acetic production by mycorrhizal fungi determined by gas chromatography- mass spectrometry. New Phytol. 94: 401-404.

Hansen.J. (1987). Stock plant lighting and adventitious root formation. HortScience 22 (5): 746-748.

Harris,G.A. and Campbell,G.S.(1989). Automated quantification of roots using a simple image analyzer. Agronomy Journal (Nov.1989, in print).

Hartmann.H.T. and Kester.D.E. (1985). Plant propagation: Principles and practices. Prentice-Hall Inc., Englewood Cliffs, New Jersey. 622 pp.

Howard,B.H. (1968). The influence of IBA and basal temperature on rooting of apple rootstock hardwood cuttings. J. hort. Sci. 43, 23-31.

Howard,B.H. and Nahlawi.N. (1970). Dipping depth as a factor in the treatment of hardwood cuttings with IBA. E. Mailing Res. Stn. R. 1969.

Keane.P.J.; Kerr.A. and New, P.B. (1970). Crown gall of stone fruit II. Identification and nomenclature of Agrobacterium isolates. Aust. J. biol. Sci. 23:585-95.

Keen, N.T. (1981). Evaluation of the role of phytoalexins. In Plant Disease Control, Staples, R.C. ed., J. Wiley & Sons, New York, pp. 155-177.

39

Klee.H.; Horsch.R. and Rogers,S. (1987). Aprobacterium-mediated plant transformation and its further implications to plant biology. Ann. Rev. Plant Physiol. 38: 467-486.

Leyval.C. and Berthelin.J. (1988). Interactions between ectomycorrhizal fungi and phosphate-solubilizing bacteria: Phosphorus mobilization from different inorganic phosphates. In: Proc. of the 7th Intntl. Symp. on Environmental Biogeochemistry. Viterbo, Italy, Sept. 1985 (in press).

Linderman.R.G. (1986). Mycorrhizal interactions with the rhizosphere microflora: the mycorrhizosphere effect. Phytopathology 78 (3), 366-371.

Linderman.R.G. and Call.C. (1977). Enhanced rooting of woody cuttings by mycorrhizal fungi. J. Am. Soc. Hortic. Sci.102 (5): 629-632.

Linderman.R.G. and Paulitz.T.C. (1989). Mycorrhizal- rhizobacterial interactions. In: Biological control of Soil-borne Plant Pathogens. Hornby,D.; Cook.R.J.; Henis.Y.; Ko.W.H.; Rovira.A.D.; Schippers.B. and Scott,P.R. (eds.). CAB International (in press).

Madej.A. and Haggblom.P. (1985). Radioinmunoasssay for determination of indole-3-acetic acid in fungi and plants. Physiol. Plantarum 64: 389-392.

Marini.R.P. (1983). Rooting of hardwood cuttings as affected by shoot position and thickness. HortScience 18 (5): 718-719.

Moore.L.; Aichele.M.D.; Millikan.D.F. and Johnson,H.G. (1980). Reevaluating hairy root disease. Amer. Nurseryman, Jan.: 10-11 and 116-117.

Moore,L.; Warren,G. and Strobel.G. (1979). Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterium rhizogenes. Plasmid 2, 617-626.

Nelson,S.D. and Clough.K.S. (1989). Rooting, growth and infection of Cotoneaster and Cornus cuttings by three Glomus species. In Mycorrhizae in the next decade; eds. Sylvia,D.M., Hung.L.L. and Graham,J.H. U. of Florida. P. 297.

Phillips,J.M. and Hayman.D.S. (1970). Improved procedure for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans, of the Brit. Mycol. Soc. 55, 158-161.

Powell,C.L. and Santhanakrishnan.P. (1985). Effect of mycorrhizal inoculation and phosphorus fertilizer on the growth of hardwood

40

cuttings of kiwifruit (Actinidia deliciosa cv. Hayward) in containers. New Zealand J. of Agric. Research, 29:263-268.

Raj, J.; Bagyaraj,D.J. and Manjunath.A. (1981). Influence of soil inoculation withn vesicular-arbuscular mycorrhizae and a phosphate-dissolving bacterium on plant growth and P32-uptake. Soil Biol. and Biochem., 13, 105-108.

Rouillon.R.; Gay.G.; Bernillon,J.; Favre-Bonin,J. and Bruchet.G. (1985). Analysis by HPLC - mass spectrometry of the indole compounds released by the ectomycorrhizal fungus Hebeloma hiemale in pure culture. Can. J. Bot. 64: 1893-1897.

Strobel,G.A. and Nachmias.A. (1985). Aprobacterium rhizopenes promotes the initial growth of bare rootstock almond. J. of Gen. Microbiol. 131, 1245-1249.

Strobel.G.A.; Nachmias.A. and Hess.W.M. (1987). Improvements in the growth and yield of olive trees by transformation with the Ri plasmid of Aprobacterium rhizopenes. Can. J. Bot. 66: 2581-2585.

Suit, F.R. (1933). Pseudomonas rhizopenes (R.B.W.K. and S.); its host relations and characteristics. Iowa State Coll. J. Sci. 8: 131-173.

Tepfer.D. (1984). Transformation of several species of higher plants by Aprobacterium rhizopenes: sexual transmission of the transformed genotype and phenotype. Cell 37, 959-967.

Verkade,S.D. (1987). Effect of Glomus fasciculatum on rooting of Viburnum dentatum during propagation. In Mycorrhizae in the next decade; eds. Sylvia,D.M., Hung.L.L. and Graham,J.. U. of Florida, p. 298.

Westwood.M.N. (1982). Rootstocks for pear. Proc. of Oregon Hort. Soc. 73, 64-79.

Westwood.M.N. and Brooks, R.M. (1963). Propagation of hardwood pear cuttings. Proc. Intnl. PI. Prop. Soc. 13, 261-268.

Yoshikawa.M.; Gemma,H.; Sobajima.Y. andMasago.H. (1986). Rooting cofactor activity of plant phytoalexins. Plant Physiol. 82, 864-866.

Zambryski,P.; Tempe.J. and Schell.J. (1989). Transfer and function of T-DNA genes from Aprobacterium Ti and Ri plasmids in plants. Cell 56: 193-201.

V. APPENDIX

A)

41

TREATMENTS PARAMETER

ROOTING AGROB. COMPOUND PHIZ.

CONTROL CONTROL ■■ A4 " A47B3 " TRIOS

IEA 2500 CONTROL .. A4 " A47S3

TR105 ISA 5000 CONTROL

" A4 " A47e3

TR 1 05 POWDER CONTROL

.. A-;

.. A47S3 " TR105

ROOT NUMBER

(.) 25 0 S5 0 46 i 29 0 58 1 38 rt 78 4 94 0 25 0 85 0 4£, 1 29 0 58 1 38 2 71 4 94 7 21 7 25 ^ 21 6 89 3 17 4 48 7 04 6 06 4 50 3 71 4 17 5 08 i, 13 4 42 3 21 o 20

ROOT LENGTH

MEAN ST D

8 00 0 00 S 00 1 00

11 00 4 36 6 18 1 60 o 68 2 45 8 38 2 09 o 50 3 26

10 77 2 84 10 06 2 69 9 47 1 69 o 92 1 S3

10 68 3 42 10 21 2 32 12 79 4 95 11 79 2 2B 16 67 17 IS

ROOT BRANCH.

20. 00 6. 67

10. 00

1 4 . 38 19. 44 14.71 22.78 24. 00 19. 17 28.42 17.37 40.71 32. 11 16.67

12 19-.

15

0/ 47

IS

B)


ROOTING AGROB. COMPOUND RHIZ.

CONTPCL

IBA 2500

POWDER

CONTROL A4

A47S3 TR 105

CONTROL A4

A47S3 TR105

CONTROL A 4

A47S3 TR 1 05

CONTROL A4

A4783 . TR105

ROOT NUMBER

ME :AN ST D

0 00 0 00 1

— — o ■?-?

0 88 1 4S 13

4 25 4 48 4 03 4 95 5 17 4 67 5 96 4 S5 5 83 6 79 3 67 4 25 6 75 6 6° ■7 46 5 80 2 00 2 59 1 79 2 30 2 83 3 98 I 42 2 17

ROOT LENGTH

MEAN ST D

0 00 0 00 7 50 1 90 7 50 2 00 8 14 0 69 10 "70 3 89 9 87 3 02

10 50 3 50 10 19 2 66 10 67 1 99 o 43 2 98 10 12 1 41 11 53 2 e>5 14 27 3 ,"*'.'? 11. 08 3 55 o 60 2 46 o^ •?2 1 92

ROOT BRANCH.

JT D

6 00 5 I £.

1 2 50 17 53 7 14 4 SS

36 43 "^S 72 30 00 21 7 i

35 00 25 -73

r«3 33 20 OS 26 67 23 81 13 57 23 4 1 24 71 15 46 39 47 29 34 41 o^ 37 37

15 S3 15 64 23 00 24 52 17 78 10 93

Table 1. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on root numbers, root length, and % of root branching of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.

A)

42


ROOT INC- AGRCB. ROOT NUMBER ROOT LENGTH ROOT BRANCH. COMPOUND RHI 2 .

MEAN ST P MEAN ST D MEAN ST D

CONTROL CONTROL 0.54 1.4! 10.75 2. £3 7. 50 0. 57 A4 25 2 1 1 7 90 1 91 10.00 21 60

" A47S3 1 i7 2 37 E 00 2 45 15.00 i 7 SO " TR105 3 Oi 3 SI 7 £3 -? 91 9.47 13 53

IBA 2500 CONTROL . 7 21 5. 82 o 91 4 43 1 7. 39 18 15 „ M £ 33 6 55 7 94 2 33 IS.24 9 51 .. A47e- i. 00 5 75 e 44 2 £0 22.78 IS .'tO

.. TR105 Z I 3 3 58 9 63 1 99 23.64 28 17 IBA SOOO CONTROL 10 13 7 87 o 77 2 83 25. 00 1 £ 2£

•i A 4 o 04 10 74 S 55 1 99 IS. 50 IS 43 " A4783 "7 25 4. 87 o 27 3 71 17. 39 IS 15

TR 1 05 g 04 £ 08 10 47 2 34 4 1 . OS 24 24 POWDER CONTROL E 33 c o.t o 58 1 77 38. 95 28 65

AJ S 54 o 27 S 61 2 6'= 31.67 20 65 ., A47S3. o 96 e 03 o 35 1 76 24.00 1 6 36 „ TR 105 5 33 n 76 o 61 -? 68 27. S3 25 04

B)


ROOTING AGR03. ROOT NUMB ER- ROOT LENGTH ROOT BRANCH. COMPOUND RHI2.

MEAN ST D MEAN ST D MEAN ST D

CONTROL CONTROL 0.46 1 . 38 8. 33 1.53 13.33 5.77 " A4 I or- -2 95 o 00 2 87 £.67 7 07 " A47S3 3 75 *=: 94 9 oc. 4 24 20.00 24 83 " TR 105 2 63 3 35 o ^c- 3 05 10.71 12 07

IBA 2500 CONTROL 5 I "T ~ 48 10 75 1 97 31 . 50 27 00 „ A4 8 — w1 y 33 10 59 3 89 37. 90 24 £3 „ ■ ,. A4783 7 21 7 33 1 x ■< o 5 47 31.43 22 £5

TRIOS 6 S3 6 41 1 1 22 2 36 36. £7 22 49 IBA 5000 CONTROL 4 13 5 07 11 36 2 4" 40.00 23 21

„ A4 10 i 3 9 r?9 10 50 3 14 72. 27 -?.;-, 4 6 ,. A47S3 6 54 6 42 9 90 2 75 22. 11 15 i ">

„ TR105 3 3^ o 35 10 21 I 25 '"?9. ^^ 1 O 5° POWDER CONTROL *"-" 75 5 £1 1 '■"' 9 3 2 52 33.75 25 00

„ A4 5 50 i 23 1 *■> 00 2 9° 4t=. 29 "9 21 " A4783 5 -70 3 82 1 1 47 2 17 29.47 '-'O 94

TR 1 05 A 92 A -7£ 11 00 2 i^. 35.29 27 39

Table 2. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on root numbers, root length, and % of root branching of mist propagated hardwood cutting of OHXF 282, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.

43

A)

TREATMENTS

ROOT I t-JG COMPOUND

A5R0B. RHIZ.

JURVU'AL

MEAN ST D

PARAMETER

ROOTING RATE.

MEAN

ROOT DRY WT.

MEAN 5T D

CONTROL CONTROL 0.03 0 17 1 96 0 -7^. 0.01 0 02 " A4 0. 2? 0 4S 2 21 1 06 0. 02 0 03

A47S~ 0.46 0 09 2 46 I 10 0. 04 0 03 '• TRIOS 0.75 0 rt i ~ i y. 0 99 0.05 0 03

I BA CS'l"'.' CONTROL 0.71 0 37 ~. 63 0 82 0. 16 0 18 .. A4 0.67 0 27 3 04 1 40 0. 12 0 1 1

A47S3 0. 7? 0 25 3 46 1 02 0. 13 0 16 TRIOS- O. 79 0 OS 3 38 1 10 0.12 0 13

IBA 5000 CONTROL 0.75 0 09 3 oo 1 27 0. 13 0 1 2 " A4 0. 62 0 ■TC-. 2 92 1 44 0. 10 0 1 1

A47S3 0.54 0 37 2 58 ; 50 O.OS 0 10 TR 1 OS 0.67 0 36 3 50 i 06 0. 19 0 19

F'OWDER CONTROL 0.7"? 0 16 3 33 1 "^5 0.12 0 IS " H4 0. 75 0 21 3 OS 1 25 0. IS 0 20

A47B.7. 0.79 0 OS 3 46 1 10 0.21 0 IS TR! 05 0.63 0 25 3 21 1 14 0. 11 0 15

B) TREATMENTS

ROOT INS AGROB. COMPOUND RHIZ.

SURVIVAL 7.

PARAMETER

ROOTING RATE. ROOT DRY WT.

CONTROL 0 00 0 00 A 4 Q 63 0 21

AH 783 0 46 0 16 TRIOS- 0 46 0 1 £.

CONTROL ,-) 54 0 28 A*1 0 i T 0 44

A4 7S3 0 S3. 0 34 TRIOS 0 87 0 09

CONTROL 0 £.•3 0 37 A 4 0 63 0 •70

A^783. 0 79 0 16 TRIOS 0 79 0 16

CONTROL 0 4 6 0 23 A4 r> SO 0 20

A4 7S3 0 46 0 25 TP105 0 54 0 25

1 91

79 58

0.

1

1.

^ 04 or- 1.

3 50 0. 3 71 0. 3 13 1.

3 29 1.

->

2

46 58 ■7^.

67

1

1. 1.

2 63 1.

10 r> 04 0 06 14 0 04 05 23 0 1 -3 0 1 9 44 0 14 0 1 6 98 0 17 0 1 7

S6 0 16 0 14 1 9 0 12 0 14 29 0 08 0 10 23 0 14 0 1 1 10 0 23 0 20 38 0 12 0 20 3-3 0 OS 0 15 24 0 03 0 1.3 28 0 It 0 49

Table 3. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on survival index, rooting rating, and root dry weight of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.

A)

44

B)


ROOT INS ASRCB. COMPOUND RHII.

CONTROL CONTROL " A4 •■ A47e3

TR 1 05 IBA 2500 CONTROL

,. A4 A47S~

.. TR105 IBA 5000 CONTROL

Afl „ A47S; „ TRIGS

POWDER .CONTROL At

A47S3 " TR105

MEA!. ST D

0. 21 0. 17 0.34 0. 14 O. 09 0. 1 4

0. 17 O. 16 O. 17 0. 24 O. 32 0. 21 0. 1 4

ROOTING RATE.

ST D

04 1 04 54 1 32 83 1 09 54 0 °S SS 0 61 29 1 20 76 1 i -?

75 0 85 S3 0 57 53 1 02 75 0 85 50 1 06 4fc 1 14 33 1 20 50 1 14 88 0 61

ROOT DRY WT.

MEAN

0. 01 0 02 0. 03 0 06 0. 04 0 05 0. 07 rjp OS 0 09 0 05 0. 07 0 07 0 09 0 '07 0. 12 0 i: 0 1 1 0 08 0. 1 1 0 1"?

0 12 0 14 0. 16 0 11 0. 14 0 12 0. 1 1 0 1 i

0 14 0 14

TREATMENTS

ROOT ING COMPOUND

AGROB. RHII.

ci ipi; T \ ;<:

MEAI.'

iL :-:

ST D

PARAMETER-

ROOTING RATG.

MEAN ST D

ROOT DRY WT.

MEAN

::ONTPC'L

IBA 2500

IBA 5000

POWDER

CONTROL A 4

A47Sr. TR 1 05

CONTROL H4

A47E3 TR105

CONTROL H4

A47S3 TR 105

CONTROL A4

A4783 TR105

0. 35 0.34

0.14 0. 1 4

O. 40 0. 1 o

0. 40 0. 1 6 0. 29 0. 1 6 0. 03

13

SS

. SO

54 i 06 58 0 88 67

1 21 0 99 79 0 72

oc

88 1 42 00 1 35 88 1 39 38 1 14 — * 1 29

0 01 0. 02 0 03 0. 05 0 09 0. 13 0 07 0. 11 0 12 0. 10 o. 16 0. 1 ~ 0 15 0. 13

0 16 0. 13 0 12 0. 14 0 22 0. 1 6 0 10 0 10 0 16 0. 17 (•-. 12 0 14 0 16 0. 1 9 0 15 0 13 0 14 0. 13

Table 4. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on survival index, rooting rating, and root dry weight of nist propagated hardwood cuttings of OKXF 282, A) no VAM, B) VAN.. Each mean represents the mean of 24 cuttings.

45

A)

B)

TF.EATMEWTS PARAMETER

F.OOTINE A6F.0B. ROOT AREA SKOOT NUMBER SHOOT DP- t' WT. COMPOUND RHIZ.

MEAN ST D MEAN ST D MEAN ST r

CONTROL CONTROL 2. 17 5. 13 0. 13 0.4 5 0.08 0.27 H4 1 48 7 O "* 0.4 2 0. 53 0. 24 ','.■ 72

A47S~ 3. 91 c 07 0.63 0. 77 0. 25 0 ~i

" TR1C'5 1 1 73 12.30 0.53 0.83 0. 39 0 7■'~,

IBA 2500 CONTROL 4? ~~ 24. 50 0. 96 0.69 6. 17 i. ^.'w

A4 32 i y 21.74 1. 04 1. 12 5. 33 -7 ■09 " A4 7S7 30 34 23. 25 0.96 0.69 0.65 0 51 .. TR105 28 f:2 23.48 0.96 I . 00 0.63 0 4 4

IBA SOOO CONTROL 27 67 22. 40 1.54 1.22 9.67 7 64 H4 24 25 24. 43 0.83 0.76 0.66 0 ~C

.. A47P.3 10 00 15.36 0.S3 0.92 0.59 0 55 TRI05 41 50 28. 27 1. 33 1 . 09 0.99 0 5"

POWDER CONTROL IS 58 16. 27 1.13 0. 95 0. 66 o ■a.i

A4 4 0 —— 24. 13 0.33 0. S7 0. 63 0 5° ,. A47S-7 33 ?e 26.41 0. 3S 0. 74 0.6° 0 52

TF:105 12. 57 15.20 1.13 0.95 0. 53 0 4 ^-

TREATMENT PARAMETER-

ROOT IM5 AC-ROB. ROOT AREA SHOOT NUMBER SHOOT DRY WT. COMPOUND RHIZ.


CONTROL CONTROL 0. 00 0.00 0 08 0.41 0.84 0 a i

ft 4 o 20 9.02 0 59 O.SS 0 36 0 79 -.■3 7?- 8 73 10.37 0 50 0. 59 0 4 7 0 49

•■ TPIO'5 5 23 7.67 0 50 0.S3 0 34 0 J0 IBA 2500 CONTROL •31 25 27.71 0 9"* 0.93 0 59 0 54

A4 29 67 24. 69 0 83 0. 74 Oi 53 0 52 " A47S3 29 10 28.24 1 08 0.86 0 80 0 53

TR105 27 58 16. 18 1 46 1. 06 0 96 0 57 IBA 5000 CONTROL 18 42 28.04 1 04 1.08 0 77 0 65

„ A4 1 3 02 18.57 0 92 0.93 o 72 0 7.2 " A47E-. 27 17 24. 00 1 25 1 . 07 0 77 0 4 9

TRIOC 4 3 59 29. 72 1 25 0. 99 0 91 0 66 c'OWDER' CONTF;^" 30 7.7 33. 18 0 58 0. 77 0 47 0 55

„ A 4 16 42 23.49 0 63 0.83 0 33 0 42 .. A4783 7 75 17.67 0 75 0. 99 0 43 0 63 " TR105 12 62 19.95 0 75 1 . 03 0 36 0 J7

Table 5 Effect of VA mycorrhizas, Agrobacterium rhizogenes, and rooting'compounds on root area, shoot number, and shoot dry weight of mist propagated hardwood cuttings of OHXF 217, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.

46

A)

TREATMENTS PARAMETER-

ROOT ING AGROB. ROOT AREA SHOOT NUMBER SHOOT DF Y WT. CONFOUND RHI Z .


CONTROL CONTROL 0.58 2. 02 0.33 0. 70 0.21 0.36 " A 4 4 98 7. 61 67 0. 92 0. 19 0.28

64787- 8 38 9. 86 0 67 0.96 0. 1 8 0.27 " TR1C5 S S3 S.96 79 0.78 0. 32 0.2S

IBA 2500 CONTROL 17 45 9.42 67 0.87 0.67 0.29 .. A4 < e- 58 12.87 29 1 . 04 0.56 0. 50

A47S3 IS 0£ 12.54 25 0.85 0. 69 0.50 TR105 15 57 10. 40 33 0.82 0. 65 0.4 1

IBA 5000 CONTROL 24 58 14.13 29 0. 86 0. 67 0.33 A4 24 li. 21. 18 54 1. 02 0. 62 0.44

.. A47E7 21 18 18.77 42 0.83 0.61 0.3S TRIOS- 32 58 12. 18 08 0.93 0. 59 0.47

c-OWDER CONTROL 27 OT 2~. 1 6 08 0.88 0.65 0.46 „ A4 20 90 15. 83 96 0.86 0. 57 0. 44 " A4787- ~*3 42 24.72 -7S 1.11 0. 72 0.41

TR10S 1 ? 16 15.24 21 0.83 0. 58 0. 32

B)

TREATMENTS- PARAMETER

ROOT !N6 AGROB. ROOT AREA SHOOT NUMBER SHOOT DRY WT. r-nMC-Q' !M[} F;KI Z.


ni--' IT=0L CONTROL 1.42 4.91 0 08 0.28 0. 09 0.31 ■' H4 4 66 6 87 0 50 0.72 0. 20 0. 2"'

A4 7S7 17 92 i p. 03 0 42 0. 72 0. 23 0. 27 7R105 o 68 14 70 0 92 1. 14 0.38 0. 40

IBA 2500 CONTROL 24 46 14 05 1 38 0.97 0.74 0.49 „ A4 ~o 67 21 93 1 38 0.97 0. 72 0. 46 ., A4783 25 S3 15 45 1 33 1.01 0.59 0.39

TRIOS- 33 75 19 86 1 29 1.12 0.S8 0.58 ;BA 5000 CONTROL 27 76 "^3 5-4 0 92 0.97 0.51 0.49

.. A4 4 0 92 19 32 1 46 1. 06 0.72 0.44 A47S7 18 72 18 23 0 88 0.74 0.50 0. 45

„ TRIOS 38 73 28 24 1 04 1. 04 0.60 0. 52 FOWDER CONTROL 21 08 22 93 0 9'*1 1. 02 0. 56 0.62

.. A4 33 23 23 76 0 96 1. 12 0. 60 0.61 " A4787. 26 58 21 53 1 04 0.91 0.64 0.4! „ TR105 22 00 18 4 5 0 02 0.93 0.41 0.34

Table 6. Effect of VA mycorrhizae, Agrobacterium rhizogenes, and rooting compounds on root area, shoot number, and shoot dry weight of mist propagated hardwood cuttings of OHXF 282, A) no VAM, B) VAM. Each mean represents the mean of 24 cuttings.

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AN ABSTRACT OF THE THESIS OF MYCQRRHIZAE. …