Vegetative Propagation Techniques for Oak, Ash,Sycamore and Spruce

David ThompsonFiona Harrington

Gerry Douglas

Michael J. HennertyNasrin Nakhshab

Roger Long

iii ii

COFORD, National Council for Forest Research and DevelopmentAgriculture BuildingUniversity College DublinBelfield, Dublin 4IrelandTel: + 353 1 7167700Fax: + 353 1 7161180© COFORD 2001

First published in 2001 by COFORD, National Council for Forest Research and Development, UniversityCollege Dublin, Belfield, Dublin 4, Ireland.

All rights reserved. No part of this publication may be reproduced, or stored in a retrieval system ortransmitted in any form or by any means, electronic electrostatic, magnetic tape, mechanical, photocopying,recording or otherwise, without prior permission in writing from COFORD

ISBN 1 902696 19 0

Title: Vegetative Propagation Techniques for Oak, Ash, Sycamore and SpruceAuthors: David Thompson, Fiona Harrington, Gerry Douglas, Michael J. Hennerty, Nasrin Nakhshab andRoger Long

Citation: Thompson, D., Harrington, F., Douglas, G., Hennerty, M. J., Nakhshab, N. and Long, R. 2001.Vegetative Propagation Techniques for Oak, Ash, Sycamore and Spruce. COFORD, Dublin.

The views and opinions expressed in this publication belong to the authors' alone and do not necessarilyreflect those of COFORD.

Production: Magner Communications

TABLE OF CONTENTS

ACKNOWLEDGEMENTS vi

FOREWORD 1

PART I: SOMATIC EMBRYOGENESIS IN SITKA SPRUCE AND OAK

SUMMARY 2

1. INTRODUCTION 3

2. SITKA SPRUCE SOMATIC EMBRYOGENESIS 4

2.1 Materials and methods/results 4

2.1.1 Initiation 4

2.1.2 Maintenance 6

2.1.3 Plantlet production 6

2.1.4 Embryogenic suspension cultures 10

2.1.5 Production costs 10

2.1.6 Plantlet quality 11

2.1.7 Field trials 12

2.2 Conclusions 12

3. OAK SOMATIC EMBRYOGENESIS 13

3.1 Materials and methods/results 13

3.1.1 Initiation 13

3.1.2 Maintenance 13

3.1.3 Maturation/proliferation and germination 13

3.2 Conclusions 14

4. OVERALL CONCLUSIONS REGARDING SOMATIC EMBRYOGENESIS 15

5. REFERENCES 15

PART II: VEGETATIVE PROPAGATION OF SELECTED REPRODUCTIVESTOCKS OF ASH AND SYCAMORE

SUMMARY 16

1. INTRODUCTION 17

2. MATERIALS AND METHODS 18

3. RESULTS AND DISCUSSION 18

3.1 Rooting in cuttings derived from seedlings and micropropagated plants 18

3.2.2.4 Length of roots 39

3.2.2.5 Root fresh weight 39

3.2.2.6 Shoot growth (height) 39

3.2.2.7 Shoot fresh weight 40

3.2.2.8 Discussion of the effect of CO2enrichment, basal media, conductivity level and

supporting matrix on photoautotrophic growth of ash explants in vitro 40

3.2.3 Effect of kinetin and NAA on rooting, bud break and shoot growth of ash cuttings 41

3.2.4 Effect of BAP and NAA on bud break and shoot growth of ash cuttings 41

3.2.5 Effect of auxin on rooting and shoot growth of ash semi-hardwood cuttings 41

3.2.6 Effect of hormone and type of cuttings on bud break and shoot growth of ash 41

3.2.7 Effect of TDZ, BAP and GA3on bud break and shoot growth of dormant sycamore buds 44

3.2.8 Establishment of in vitro stool beds 44

3.2.9 General discussion of shoot growth and multiplication in ash and sycamore 43

3.3 Rooting and weaning 49

3.3.1 Effects of IBA and NAA on rooting of ash softwood cuttings 49

3.3.2 Weaning of micropropagated material of ash and sycamore 49

4. CONCLUSIONS 50

5. REFERENCES 51

APPENDIX I 53

APPENDIX II 54

viv

3.2 Rooting of ash and sycamore cuttings from coppiced hedges 20

3.3 Cuttings from grafted elite clones of ash 21

3.4 Cuttings from grafted and self rooted elite clones of sycamore 21

3.5 Micropropagation of sycamore and ash 22

4. CONCLUSIONS 27

5. REFERENCES 28

PART III: PHOTOAUTOTROPHIC MICROPROPAGATION OF ASH AND SYCAMORE

SUMMARY 29

1. INTRODUCTION 30

2. MATERIALS AND METHODS 31

2.1 Medium preparation and use 31

2.2 Growth room conditions 31

2.3 Establishment and culture of ash seed in vitro 31

2.4 Establishment and culture of dry seed of sycamore in vitro 31

2.5 Establishment and culture of fresh seed of sycamore and ash in vitro under enriched

carbon dioxide conditions 31

2.6 Direct establishment of sycamore softwood and semi-hardwood cuttings under

enriched carbon dioxide conditions 32

2.7 Production of epicormic shoots from mature ash and sycamore 32

2.8 Experimental designs and statistical analyses 32

3. EXPERIMENTAL WORK: RESULTS AND DISCUSSION 33

3.1 Establishment of in vitro cultures 33

3.1.1 Seed germination under mixotrophic conditions 33

3.1.2 Seed germination under photoautotrophic conditions 33

3.1.3 Direct establishment of sycamore semi-hardwood cuttings under autotrophic conditions 33

3.1.4 Production and culture of epicormic shoots from mature ash and sycamore 34

3.1.5 Disinfection procedures 35

3.1.6 General discussion on the establishment phase 35

3.2 Shoot growth and multiplication of ash and sycamore in vitro 36

3.2.1 Effect of medium, BAP and activated charcoal on shoot growth and multiplication rate

of ash and sycamore nodal explants 37

3.2.2 Effects of CO2enrichment, basal media, conductivity levels and supporting matrix on

photoautotrophic growth of ash explants in vitro 38

3.2.2.1 Survival rate 38

3.2.2.2 Rooting of ash nodal explants 38

3.2.2.3 Number of roots per plant 39

vi 1

FOREWORD

Irish forest nurseries produce 80 million plants each year. Conifers account for the bulk of production - 67million plants - but the proportion of broadleaves is ever increasing and they account for the balance of 13million plants. Growing for the Future – the government’s strategy for the development of the forestrysector has set a target afforestation rate of 20,000 hectares each year to 2030, 30% of which will becomprised of broadleaves. It is vital that the programme is underpinned by the use of quality, vigorousplanting stock.

Planting stock quality contributes significantly to the return on the forestry investment. The investigationof techniques to improve planting stock quality has been an integral part of the COFORD R&D programmesince 1995. Vegetative propagation using rooted cuttings is growing in popularity as a means to rapidlyintroduce genetically improved planting stock. Other related techniques are being developed which mayhave application in both coniferous and broadleaved species. These include somatic embryogenesis andphotoautotrophic micropropagation. Both are at the early stages of development but they offer considerableadvantages in rapidly deploying superior genetic material.

COFORD has funded research on vegetative propagation in a number of projects since 1995. Under theprogramme, methodologies for producing quality planting stock material have been refined and furtherdeveloped. While further work is necessary to improve the cost-efficiency of such methods, superiorplanting stock is now being made ready for field testing under the current COFORD programme. This phasewill demonstrate how successful the development work has been.

In conclusion I would like to acknowledge the dedication and commitment of the teams of researchersinvolved in the projects reported here. The results of the research will be of considerable interest to theforest nursery sector now and into the future.

David Nevins Chairman

November 2001

ACKNOWLEDGEMENTS

Thanks are due to the following for assistance in the conduct of the experiments and the preparation of the text:Mr John Fennessy (Coillte), Mr John McNamara (Teagasc) and Mr Tom Moore (Dept. of Crop Science,Horticulture and Forestry, U.C.D).

3

1. INTRODUCTION

The irregularity associated with the periodicity offlowering and seed production in the breeding andproduction of genetically improved materialresults in significant delays in putting the results oftree breeding efforts into practical use. Vegetativepropagation techniques such as rooting of cuttings,grafting, air layering and micropropagation offer away to avoid these delays. In vegetativepropagation the cost of producing plants is critical.Because forest tree seedling costs range from£0.08 (€0.10) to £0.25 (€0.30) each, it is verydifficult to produce vegetatively propagatedmaterial at competitive prices. As a result,grafting and air layering are too expensive toemploy in large-scale propagation. Rootedcuttings of forest trees typically cost two or moretimes the cost of seed propagated material becauseof the labour required. Even if every cutting roots,which is usually not the case, this produces onlyone plant. The advantage of micropropagation isthat each culture can produce an unlimited numberof plants, at least in theory.

The problem with conventional micropropagationsystems that depend on organogenesis is that theamount of labour (handling) involved inproducing the plants results in high productioncosts. In organogenesis the process involvesculture initiation, stimulation of axillary oradventitious buds, elongation of buds into shoots,excision of shoots, rooting of shoots and transferfrom culture to the greenhouse, each step requiringhandling. The cost of Monterey pine (Pinusradiata D. Don.) plants propagated byorganogenesis was estimated at between five to sixtimes the cost of conventional seedlings (Smith1991).

In somatic embryogenesis the shoot and root poleof the plant are formed in one step rather than twoseparate steps. Because it involves fewer stepsand therefore requires less labour, somaticembryogenesis could produce plants at costs thatmay be similar to rooted cutting costs or evenapproach seedling production costs. For thesereasons somatic embryogenesis was tested as apropagation method for Sitka spruce and oak.

The basic steps in the process of somaticembryogenesis are:• initiation of embryogenic cell lines;• growth and maintenance of cell line;• somatic embryo formation;• maturation of somatic embryo;• germination of somatic embryo;• conversion of somatic embryo into a plant.

For a review of the methods for production ofsomatic embryogenesis the reader is referred to areview of the conifers by Tautorus et al. (1991)and of oak by Wilhelm et al. (1999).

2

SOMATIC EMBRYOGENESIS IN SITKA SPRUCE AND OAK

DAVID THOMPSON1 FIONA HARRINGTON2

SUMMARY

Somatic embryogenesis presents a potential method for the rapid propagation of Sitka spruce (Piceasitchensis (Bong.) Carr.) and oak (Quercus species). In Sitka spruce, initiation of embryogenic cell lines isthe main bottleneck (about 4% of the embryos cultured formed embryogenic cell lines) and all modificationsof the initiation process have failed to significantly increase initiation rates. However, once embryogeniccell lines are established, somatic embryos can be formed, matured, germinated and converted into completeplants. A preliminary analysis of the estimated production costs has identified the handling of individualplants as the most costly step in the process.

In oak the situation is slightly different because while initiation rates can be up to 27%, it is the switchingoff of the production of somatic embryos that presents the greatest bottleneck.

Nevertheless, both systems, even in their present state of development, are capable of producing smallnumbers of plantlets for establishment in field trials. Further work is necessary to improve the efficiency ofthe process and to prove the genetic and physiological uniformity and quality of the plant material produced.Larger field trials are needed to demonstrate the performance of material propagated by this method.

1 Coillte Research Laboratory, Newtownmountkennedy, Co Wicklow, (david.thompson@coillte.ie)2 Coillte Research Laboratory, Newtownmountkennedy, Co Wicklow, (fiona.harrington@coillte.ie)

5

Embryos from five commercial seed lots wereinitiated in culture to compare initiation frequencywith that of selected families (Table 2). Initiationfrequencies ranged from 0.6 to 4%, which weresimilar to results obtained with selected seeds inthe first year of the project.

A range of experiments, with the specific aim ofimproving initiation frequency from matureembryos, was carried out. The standard initiationmedium with various modifications was used.Twenty-five embryos from each seed source wereused for each treatment.

The effect of carbohydrate source andconcentration was tested (Table 3). Replacingsucrose with maltose resulted in the developmentof adventitious shoots. These shoots weretransferred to MS medium where they continuedto develop. Sucrose concentrations of 1 to 4%were tested. The best treatment was 1% sucrose(10 g L-1).

The use of activated charcoal (1.25 g L-1) in theinitiation medium stimulated embryo germination rather than embryogenesis. All roots, hypocotylsand cotyledons were transferred back to freshinitiation medium to try to induce embryogeniccultures. None were obtained (data not presented).

Various concentrations of the auxins 2,4-D (0.5 to16 mg L-1), NAA (0.5 to 6.0 mg L-1) and Picloram(0.25 to 2.0 mg L-1) were tested for their ability toinitiate embryogenic tissue (Table 4). Cultureswere successfully initiated with 2,4-D at 2.0 mg L-1 in one of the two families tested. Higherinitiation frequencies were obtained with NAA.The optimum concentrations of NAA were in therange of 1.0 to 4.0 mg L-1.

The effect of gelling agent and concentration wastested (agar at 7, 10, 15 g L-1 and phytagel at 2, 4,6, 8 g L-1). No effect on initiation was observed(data not presented).

The effect of varying the concentrations of NH4Cl

and NaNO3is known to influence the formation of

embryogenic tissue in other plant species. Trialswith Sitka spruce resulted in embryogenic culturesdeveloping at both high and low concentrations ofnitrate (Table 5). Further work on the effect ofnitrate, ammonium and total nitrogenconcentration on somatic embryogenesis needs tobe carried out.

A medium, (BLG medium, Brown and Lawranceglutamate), in which the inorganic ammoniumnitrogen is replaced by an equivalent amount oforganic nitrogen in the form of glutamine wastested. A high initiation frequency was reported to

Family Number ofembryoscultured

Sterileseeds in culture

%

Embryoswith callus

development%

Number of embryogenic

cultures

Initiation frequency

%

V130V131W62W17SQ

200150100100100

8486989650

100100100100100

13132

0.62.31.03.14.0

TABLE 2: INITIATION OF EMBRYOGENIC CULTURES FROM FIVE COMMERCIAL SEED LOTS.

TABLE 3: EFFECT OF CARBOHYDRATE TYPE AND CONCENTRATION ON EMBRYOGENIC INITIATION(25 EMBRYOS/TREATMENT).

10 g L-1

8%8%

30 g L-1

0%0%0%0%

Sucrose574251Maltose191574587576

20 g L-1

0%0%

30 g L-1

0%0%

40 g L-1

0%0%

Treatment/family Concentration/initiation frequency

Stimulation of adventitious shoots

4

2. SITKA SPRUCE SOMATICEMBRYOGENESIS

Sitka spruce embryogenesis involves thefollowing four steps - initiation, main-tenance/proliferation, maturation and germination.

2.1 Materials and methods/results

2.1.1 Initiation

Mature seeds from progeny tested families wereused to initiate embryogenic cultures. Seeds weresurface sterilised in a 20% bleach solutioncontaining several drops of Tween 20 as a wettingagent for 20 minutes followed by three rinses insterile, distilled water. Approximately 100zygotic embryos were dissected from the seedsand were cultured on a basal induction mediumconsisting of a modified Murashige and Skoog(MS) mineral medium supplemented with 2,4-D(1.0 mg L-1), BAP (0.5 mg L-1) and kinetin (0.5 mg L-1) (Appendices I and II). The mediumcontained 30 g L-1 of sucrose and was solidifiedwith 6 g L-1 agar. All cultures were maintained inthe dark at 25 ˚C for 2 months before examination.

Embryogenic tissue in conifers is not a typicalunorganised or ‘callus’ type of tissue (Tautorus etal. 1991). It consists of two distinct, organisedcell types; the small meristematic cells of theembryogenic ‘heads’ and the large, elongatedsuspensor cells. These two cell types are verysimilar to the types of cells that form zygoticembryos in developing ovules. Thus, to refer tothe tissue that produces somatic embryos as‘callus’ or even ‘embryogenic callus’ is incorrectbecause the tissue is highly organised and consistsof very highly specialised cells. The term‘embryogenic suspensor mass’ or ESM has also

been suggested to distinguish this unique type oftissue.

In the first year of the project over 4,000 maturezygotic embryos (representing 27 selectedfamilies and a control) were used to initiateembryogenic cultures. Twelve of the familiesfailed to show any response, eight familiesdeveloped non-embryogenic callus and sevenfamilies initiated embryogenic cell lines in culture.These low rates were probably, in part, due to poorquality, ageing seed. In later work, a total of 15embryogenic cell lines were established withinduction frequencies (expressed as a percentageof the initial number of zygotic embryos cultured)ranging from 0.9 to 4.8%. Among the familiesthat formed embryogenic cell lines were families191, 574 and 587, which are some of the bestperforming families in Coillte’s Sitka spruceimprovement programme.

In another initiation study, seedling cotyledons(the first seedling leaves) were tested as an explantsource. One cotyledon was removed from eachseedling (100/family). Four families were tested(251, 574, 589 and 183). Embryogenic tissue wasobtained from only one family (589) and theinitiation frequency was very low (1%). Needleswere also removed from some plants produced bysomatic embryogenesis of family 183, but noembryogenic tissue was produced.

In the second year of the project, six of the samefamilies were again initiated in culture. In contrastto the first year’s results, only one family (574)developed embryogenic cultures (Table 1). Theviability of the seed being used was tested bysowing it in compost. Only one family (574) wasstill viable.

Family Number ofembryoscultured

Sterileseeds in culture

%

Embryoswith callus

development%

Number of embryogenic

cultures

Initiation frequency

%

191574580587589576

420400100427490400

808330923695

0680606115

020000

00.60000

TABLE 1: INITIATION OF EMBRYOGENIC CULTURES FROM MATURE ZYGOTIC EMBRYOS OF SIXSELECTED FAMILIES.

7

maintenance medium to a modified MS mediumcontaining abscisic acid (ABA) at 50 mg L-1 andactivated charcoal (1.25 g L-1). Cultures aremaintained on this medium for 2 months in thedark during which somatic embryos develop andmature. They are then transferred to a modifiedMS medium containing no exogenous growthregulators and placed in the light for somaticembryo germination and the production ofemblings. The effect of different geneticbackgrounds on the ability to produce somaticembryos can be seen from a comparison of 6different cell lines originating from four different Sitka spruce families (Table 6).

Not only were differences between families seen,but also differences between cell lines originatingfrom different zygotic embryos of the same openpollinated family (251) were observed.

Methods to improve plantlet production focusedon the composition of the maturation medium.Maturation of somatic embryos results in well

developed shoot and root apical meristems thatwill elongate simultaneously when ‘germination’occurs. Initial experiments were run testing theeffect of different osmotic treatments to improvesomatic embryo production. Various osmoticawere tested, including sorbitol (3%), mannitol (2and 4%) and polyethylene glycol (3, 6 and 9%).Five different embryogenic cell lines were tested,but none of the above treatments had anysignificant effect on embryo regeneration (data notpresented).

The effect of phytagel (a compound used tosolidify the nutrient medium similar to agar)concentration yielded some interesting results(Figure 1). Concentrations of 2 to 10 g L-1 weretested and compared to the standard treatmentcontaining 6 g L-1 agar. Six lines were tested. Theresults presented were the means of twoexperiments. Although the results do notconclusively show that one individual treatmentwas best, in almost all cases the standard treatmentproduced the fewest number of cotyledonary

Cell line Average number of somatic embryos per gram of embryogenic tissue

4.07.92.57.5

27.06.7

251H251I251J12C574A576B

TABLE 6: EFFECT OF FAMILY AND CELL LINE ON THE ABILITY OF SITKA SPRUCE EMBRYOGENIC CELLLINES TO FORM SOMATIC EMBRYOS.

FIGURE 1: THE EFFECT OF PHYTAGEL CONCENTRATION ON MATURATION OF EMBRYOS.

6

occur in Norway spruce (Picea abies (L.) Karst.)when embryos were cultured on BLG medium inthe light (Verhagen and Wann 1989). Noembryogenic cultures were obtained however,when this medium was tested with Sitka spruce(data not presented).

The embryogenic initiation rates achieved in Sitkaspruce are very low (maximum frequencyobserved in this work was 12%) and none of thetreatments tested appear to result in a significantimprovement in the initiation rate. All of the workpresented above has been done with maturezygotic embryos serving as the starting material.Perhaps through the use of immature zygoticembryos as the original explants, the frequency ofembryogenic initiation can be increased, as hasbeen demonstrated in Norway spruce.

2.1.2 Maintenance

Embryogenic cell lines were maintained on thesame nutrient medium as that used in the initiationof embryogenic cell lines. Cultures weremaintained on agar solidified media in petri dishes

in darkness at 25 ˚C and were subcultured everytwo weeks.

Cell lines established in the first year of the projectwere maintained in the above mentioned mannerand used for later experiments. Concerns aboutpossible loss of embryogenic potential over timeled to investigations of alternative ways tomaintain embryogenic cell lines. Cold storagewas one such approach. Fifteen cell lines wereplaced in a cold store (+4 ˚C). After 8 months allcultures were removed and transferred to freshmaintenance medium and placed in the growthroom. Three subcultures later, only two cell lines(251I and 251B) showed signs of growth orembryogenic tissue. Later, only line 251Isuccessfully regenerated emblings. Thus, coldstorage of embryogenic cell lines does not appearto be a viable option, at least at the temperaturesand conditions tested.

2.1.3 Plantlet production

Somatic embryos of Sitka spruce developed bytransferring embryogenic tissue from the

TABLE 4: EFFECT OF GROWTH REGULATOR TYPE AND CONCENTRATION ON INITIATION (25 EMBRYOS/TREATMENT).

TABLE 5: THE EFFECT OF VARYING CONCENTRATIONS OF NH4Cl AND NaNO

3ON THE FREQUENCY OF

EMBRYOGENIC CULTURE INITIATION (25 EMBRYOS/TREATMENT).

2,4-D251W62NAA251574V87W62Picloram574251

0.5 mg L-1

0%0%

0.5 mg L-1

0%0%0%0%

0.25 mg L-1

0%0%

1.0 mg L-1

0%0%

1.0 mg L-1

4%4%0%0%

0.5 mg L-1

0%0%

2.0 mg L-1

0%4%

2.0 mg L-1

4%0%8%0%

1.0 mg L-1

0%0%

4.0 mg L-1

0%0%

4.0 mg L-1

0%8%0%0%

2.0 mg L-1

0%0%

6.0 mg L-1

0%0%

6.0 mg L-1

0%0%0%0%

8.0 mg L-1

0%0%

12.0 mg L-1

0%0%

16.0 mg L-1

0%0%

Treatment/family Concentration/initiation frequency

Family 5741.5 mM2.5 mM5.0 mM

Family 2511.5 mM2.5 mM5.0 mM

1.5 mM12%0%0%

1.5 mM0%0%0%

2.5 mM0%0%0%

2.5 mM0%0%0%

5.0 mM0%0%12%

5.0 mM0%0%4%

7.5 mM0%0%0%

7.5 mM0%0%0%

NaNO3

NaNO3

NH4Cl

NH4Cl

9

treatments except in cell line 576B where thestandard did not perform as well.

Matured somatic embryos ‘germinate’(simultaneous elongation of the shoot and rootmeristems) and produce a normal looking plantlet.Germinated somatic embryos need to betransferred from the low light levels and highhumidity of the petri dish in the growth room to

the higher light levels and lower humidity of thegreenhouse. During this transition period theemblings become autotrophic. This process isknown as 'acclimatization'. This is accomplishedby potting the well-developed embling (with atleast 5 mm of new root and growth of the trueshoot above the cotyledons) in a 50:50 mixture ofpeat:perlite under a plastic propagating cover.After 4 to 6 weeks the humidity is gradually

FIGURE 4: THE EFFECT OF DESICCATION TREATMENT ON EMBRYO GERMINATION.

FIGURE 3: THE EFFECT OF SILVER NITRATE ON MATURATION OF EMBRYOS.

8

embryos. Results indicate that a higher number ofembryos may be obtained by using phytagelinstead of agar in the maturation medium. Inaddition, it would appear that an increase inembryo number occurs with an increase inphytagel concentration, up to 8 g L-1.

The effect of phytagel concentration in thematuration medium on subsequent germinationwas also tested. No significant difference ingermination rate was observed between embryosmatured under different phytagel concentrations(data not presented). Germination rates for celllines ranged from 0 to 20%. These results arelower than previous years and may indicate adecline in the performance of the cell lines(maintained for 3 years in culture before beingused in these experiments). No distinctdifferences in embryo morphology were observedbetween treatments, except that embryos tended tobe smaller at higher phytagel concentrations.

In a subsequent experiment, the effect of varyingthe agar concentration on maturation was tested(Figure 2). Results obtained indicated a decreasein embryo number development with increasingagar concentration. The effect of these treatmentson subsequent germination could not bedetermined because germination rates were zero.

These results show that there are fundamentaldifferences between agar and phytagel in theproduction of somatic embryos. In most work thegelling agent is assumed to have no role in the

tissue culture process, while these results illustratethat this is clearly not true in production ofembryogenic Sitka spruce cultures.

The gaseous plant growth regulator ethylene hasbeen shown to play a role in the development ofsomatic embryos. Therefore the effect of silvernitrate (an inactivator of ethylene effects) in thematuration medium was tested (Figure 3). Resultsshow that in some instances the standardmaturation medium performed best, while inothers the addition of silver nitrate was beneficial.Overall, it is felt that the inclusion of silver nitratein the maturation medium failed to improvematuration.

Work aimed at improving the germinationpotential concentrated next on subjecting embryosto various desiccating treatments (Figure 4). Thevarious treatments tested were agar 1% andsucrose 1.5% in the germination medium. Inaddition, embryos were desiccated under highhumidity using the well system. This uses a 25cell plastic multi-well plate with all of the outer 16and the central well filled half way with water.Somatic embryos were placed in each of theremaining eight wells and exposed to thistreatment for 3 weeks before being transferred tosolidified germination medium. All treatmentswere compared to the standard treatment. Resultsare presented as the mean of two experiments(Figure 4). The least effective treatment wassucrose 1.5% for all cell lines tested. There wereno significant differences between any other

FIGURE 2: THE EFFECT OF AGAR CONCENTRATION ON MATURATION OF EMBRYOS.

574 A 183 E 251 J 576 b

251 J 574 A 576 B

single germinating embryos. This is because thisis the only stage in the process where singleemblings are handled. Prior to this, only masses ofembryogenic cells and clusters of developingemblings (consisting of tens to hundreds ofsomatic embryos) are handled en mass. When itcomes to selecting out and handling individualembryos the production costs increasedramatically. Labour costs account for the largestpercentage of the total production costs. Thus, theassumption that producing large numbers ofembryogenic cells or indeed small plantlets inbioreactors (somatic embryogenesis productionsteps) will reduce plant production costs may notbe correct. When plants are removed from thebioreactor they will still have to be handledseparately, which will be the most costly step inthe process. If a reduction of production costs ofin vitro produced plant material is required, thenmethods for the automated or semi-automatedselection and handling of plantlets should bedeveloped.

A second important point is the overall cost of1,000 emblings. A cost of €331/1,000 emblings ishigh compared to traditional unimproved Sitkaspruce costing about €127/1,000, but it is morecomparable to the cost of genetically improvedrooted cuttings, which cost €235/1,000. It is alsoimportant to remember that these are based on anon-optimised production process. This cost isnot as high as some estimates of the cost ofmicropropagated trees (propagated byorganogenesis) that have been suggested {five tosix times the cost of conventional seedlings (Smith1991)}. With further research, the process cancertainly be improved. Production costs will onlydecrease to equal the cost of rooted cuttings andmay even approach the cost of producingseedlings.

2.1.6 Plantlet quality

Two embryogenic cell lines of the same familywere used to regenerate emblings for field trials.These were acclimatized in the greenhouse.Seedling controls of the same family were grownin the same containers, in the same media andunder the same greenhouse conditions as theemblings. Table 8 provides morphologicalcomparisons between the emblings and theseedlings.

There were no significant morphologicaldifferences between emblings and seedlings of thesame family except that the number of seedlingsthat had undergone a second flush during theautumn of 1998 was greater than either of the twoembling lines. Because late flushing is considereda characteristic of young plants that decreases withincreasing age, this observation would suggest thatthe emblings might have a more mature growthhabit than seedlings. In addition, the Sitkaemblings had a bluer needle colour than theseedlings, which may also be indicative of a moremature plant. Whether these differences were dueto differences in the maturation state of theemblings and seedlings and whether thesedifferences persist will need to be documented inthe field trials.

In similar morphological comparisons betweenrooted Sitka spruce cuttings with seedlingtransplants there was a statistically significantdifference in the number of branches, with thecuttings having fewer branches than the seedlings(Fennessy et al. 2000). The results presented hereof comparisons between emblings and seedlingsdo not show such a trend.

11

TABLE 7: ESTIMATED PRODUCTION COST OF THE STEPS IN THE PROCESS OF SITKA SPRUCESOMATIC EMBRYOGENESIS.

Stage of productionEmbryogenic tissue productionMaturation stepGermination stepSelect and transfer emblingsTotal in vitro embling production costsTotal in vitro costs Cost to establish emblings in the greenhouseYield sub-total (assume 90% survival)Overheads (20%)Total cost at the end of the greenhouse phaseCost to grow 1 year in containerTotal cost of emblings ready to go to the field

Cost€546/1,000 plates€411/1,000 plates€411/1,000 plates€673/1,000 plates€2,042/1,000 plates€165/1000 emblings€36/1,000 emblings€223/1,000 emblings€44/1,000 embling€268/1,000 emblings€63/1,000 emblings€331/1,000 emblings

10

reduced until the propagating cover can becompletely removed at which time the emblingscan be grown under normal greenhouseconditions. During 1997 about 1,367 plants weresuccessfully established in compost (Figure 5).The percentage of emblings surviving ranged from90 to 100%.

2.1.4 Embryogenic Suspension Cultures

Embryogenic cells can also be grown in a liquidmedium (the same composition as theinduction/maintenance medium but without agaror phytagel) on a rotary shaker to aerate the cells.Such suspension cultures can provide informationon the growth of the different types of cells(embryogenic and suspensor cells), which is moredifficult to assess in cultures maintained onsolidified media. Shake flask experiments wereset up with Sitka spruce embryogenic cell lines toanalyse cell growth characteristics, optimumsubculturing frequency and nutrient utilisation.Cells were grown for 14 days and analysed ondays 0, 3, 7, 10 and 14. Settled cell volume(SCV), fresh weight (FW), dry weight (DW), pHand embryo numbers were recorded on eachsampling day. Various technical problems wereencountered during several attempts to run thisexperiment. Therefore, while no useful resultswere produced during this project, the technique isworth further evaluation. Embryogenic cell

suspensions could be grown in a 'bioreactor' wherefresh nutrients are added periodically to producelarge volumes of embryogenic cells that could beinduced to form somatic embryos for large-scaleplantlet production.

2.1.5 Production costs

To identify the most costly steps in the somaticembryogenesis process, the current non-optimisedprocess was subjected to an economic analysis.Because of the low initiation frequency all costswere based on the use of an establishedembryogenic cell line. The production is dividedinto two phases, the in vitro and the plantlet orembling phase. The in vitro costs began with asingle culture that had to be grown to produceenough tissue to produce 1,000 plates ofembryogenic tissue. All the in vitro costs arebased per 1,000 petri plates. Once this had beenachieved and emblings had been regenerated thecosts were based on the cost to produce 1,000emblings.

All of the above estimates are based on the currentnon-optimised production method. The mainpoint of note is that the most expensive single stepis not in the in vitro somatic embryogenesisproduction steps (embryogenic tissue production,maturation or germination steps) as might beexpected, but rather in the selection and transfer of

FIGURE 5: THE NUMBER OF PLANTLETS SUCCESSFULLY TRANSFERRED TO COMPOST.

Num

ber

of p

lant

lets

13

3. OAK SOMATIC EMBRYOGENESIS

Oak somatic embryogenesis involves the samefour main steps - initiation, main-tenance/proliferation, maturation and germinationas in the case of Sitka spruce.

3.1 Materials and methods/results

3.1.1 Initiation

Immature acorns (late July) from six oak treeswere collected, surface sterilised and the embryoscultured on an induction medium consisting of aMS medium containing BAP 1.0 mg L-1, sucrose30 g L-1 and solidified with 6 g L-1 agar. Cultureswere maintained in the dark at 25 ˚C. Between 30and 50 immature zygotic embryos per tree wereestablished in culture. Initiation frequenciesranged from 0.7 to 26.2%. In total 24embryogenic cell lines were established in culture.The material used to initiate the cultures was fromphenotypically selected individuals, but none ofthese trees has been progeny tested so they are ofunproven genetic value.

In oak, somatic embryogenesis is achieved by theoriginal zygotic embryo ‘budding’ to form acluster of somatic embryos, which continue to'bud' to produce more somatic embryos. Theformation of an unorganised callus tissue or evenan organised embryogenic tissue, as in Sitkaspruce, does not occur. This technique can bedescribed as secondary somatic embryogenesis inwhich the immature zygotic embryo is induced toreplicate itself indefinitely.

The induction of oak somatic embryos frommature tree tissues or at least non-seed tissues suchas leaf of stem segments has been reported. Thistechnique would allow for the propagation ofproven superior mature oak trees. During thecourse of this project, leaf and stem explants frommicropropagated oak cultures initiated frommature trees, were cultured on a somaticembryogenesis induction medium. Sterileexplants from shoot tip micropropagated oak lineswere placed on MS medium {containing BAP(2.25 mg L-1) and NAA (1.8 mg L-1)} and placed inthe dark for 2 months. Non-embryogenic calluswas formed from both the leaf and stem segmentsbut no embryogenic tissue was ever observed inthese cultures.

3.1.2 Maintenance

Once an embryogenic culture had been initiated itwas maintained in culture on MS mediumcontaining BAP (0.2 mg L-1) and 3% sucrose. Allcell lines were maintained in the light at 25 ˚C andtransferred every 4 to 6 weeks.

3.1.3 Maturation/proliferation and germination

Maintenance of oak embryogenic cell lines wasstraightforward and presented no technicalproblems. The difficulty, however, was in theswitching off of the process of secondary somaticembryogenesis and stimulating the somaticembryos to mature and then germinate. Variousmaturation treatments were tested includingdifferent nutrient media, hormone combinationsand different desiccation methods. The mosteffective method was the use of a higher agarconcentration (1%). Increasing the agarconcentration effectively induces osmotic stress,which reportedly stops budding of somaticembryos and enhances maturation. After 5 weekson the high agar medium the embryos weretransferred either to a germination medium (basalmedium without exogenous plant growthregulators) or subjected to a partial desiccationtreatment (the same multi-well system describedfor desiccation of Sitka spruce somatic embryos).Partial desiccation also provides a trigger to stopbudding of somatic embryos and to start thematuration programme.

Somatic plants have been obtained; however, noone treatment has consistently produced a regularsupply of emblings. Seven emblings from cell line431/H9 and ten from line 2D have beenestablished in compost in the greenhouse. Inaddition emblings from cell line 2G (fiveemblings), 2D (three emblings) and 3C (threeemblings) are developing in culture. Theemblings growing in compost will be lined out inthe nursery.

2.1.7 Field trials

About 2,000 emblings from a range of familieswere acclimatized, grown in the greenhouse andexamined in field trials.

2.2 Conclusions

In Sitka spruce somatic embryogenesis, initiationof embryogenic cell lines is the main bottleneck.All attempts to increase the initiation frequencyhave not been successful. There are two waysaround this problem. First, by culturing largenumbers of embryos a small number ofembryogenic cell lines will always be initiated. Asecond approach, not tested in this work, would beto use immature zygotic embryos where, inspecies such as Norway spruce, initiation rates arehigher than with mature zygotic embryos. This isprobably the best solution to the problem of lowinitiation rates.

Development of a method to maintainembryogenic cell lines in a state where they willnot deteriorate is important. Cold storage (+4 ˚C)did not appear to be successful. Storage of cellscryogenically at liquid nitrogen temperature (-196 ˚C) has been successful with other cell lines,but it requires equipment to freeze the cells at aconstant rate (1 ˚C/minute) and a liquid nitrogenstorage container, both of which were notavailable for this study.

Somatic embryo formation does not present aproblem, provided the lines have been wellmaintained in culture. The use of ABA and thehigh humidity desiccation treatment arereasonably successful methods for the productionof mature somatic embryos capable of goodgermination rates. Germination rates (about 25%)could be further improved, but conversion rates of90% are very acceptable. Even the current non-optimised state-of-the-art process is capable ofproducing enough emblings for the establishmentof field trials to demonstrate the potential of themethod.

Although the process would have its ultimateapplication in the production of plant material foruse in the field, this is probably a number of yearsaway. The most likely immediate application ofthis technology will be the propagation of selectedfamilies for use as stock plants to produce cuttingsfor rooting. Later, with further improvements intechnology and reductions in the production costsit may be possible to produce emblings that will godirectly to the field. Encapsulation of somaticembryos in a coating could result in ‘artificialseeds’ that could be handled exactly like zygoticseeds.

12

ParameterHeight cmStem diameter mmNo. of branchesBranches/heightFlushed %

574A emblings30.103.05

11.600.380.00

574B emblings47.403.75

16.000.340.00

Seedling controls35.203.05

15.600.44

42.00

TABLE 8: MORPHOLOGICAL COMPARISONS OF EMBLINGS OF FAMILY 574 (20 PLANTS/PLANT TYPE)MEASURED AT THE END OF THE 1998 GROWING SEASON.

15

4. OVERALL CONCLUSIONSREGARDING SOMATICEMBRYOGENESIS

Based on the results of this project, somaticembryogenesis continues to have great potential asa vegetative propagation method for both Sitkaspruce and to a lesser extent oak. In both species,lack of the knowledge of the basic physiology andbiochemistry of zygotic embryo development isthe major bottleneck in the process. Nevertheless,a number of emblings of both Sitka spruce and oakhave been successfully produced. Further workshould concentrate on the genetic fidelity anduniformity over time of these emblings. Thepotential to use tissue from trees old enough tohave demonstrated their superior characteristics toestablish embryogenic cell lines could make true'clonal forestry' a reality. The uniformity andreliability of clonal planting stock could have asignificant effect on the production of qualitymaterial in plantation forests.

While further work is necessary to improvesomatic embryo maturation techniques, thesystem, at least in spruce, is well enoughunderstood to be able to produce sufficientmaterial to establish small field trials of emblingsproduced by this method. The next step is toestablish larger field trials of somatic embryos todemonstrate to foresters and the public the fieldperformance of this material. Further researchwork, specifically on improving initiation andmaturation rates as well as documenting thegenetic fidelity and uniformity of the resultingemblings is necessary. This information shouldhelp overcome the concerns that foresters and thepublic may have concerning propagation bysomatic embryogenesis.

5. REFERENCES

Fennessy, J., O'Reilly, C., Harper, C. P. andThompson, D. 2000. The morphology andseasonal changes in cold hardiness, dormancyintensity and root growth potential of rootedcuttings of Sitka spruce. Forestry 73 (5): 489-497.

Smith, D. R. 1991. Economic benefits ofvegetative propagation. In ProceedingsFRI/NZFP Forest Ltd. Ed. Miller, J. T. ClonalForestry Workshop, 1-2 May 1989, Rotorua, NewZealand, pp 158-60.

Tautorus, T. E., Fowke L. C. and Dunstan, D. I.1991. Somatic embryogenesis in conifers. Can. J.Bot. 69:1873-99.

Verhagen, S. A. and Wann, S. R. 1989. Norwayspruce somatic embryogenesis: High frequencyinitiation from light cultured mature embryos.Institute of Paper Chemistry Paper Series 287.

Wilhelm, E., Endemann, M., Hristoforoglu, K.,Prewein, C. and Tutkova, M. 1999. Somaticembryogenesis in oak (Quercus robur L.) andproduction of artificial seeds. In Proceedings ofApplications of Biotechnology to Forest Genetics.Eds. Espinel, S. and Ritter, E., BIOFOR-99, 22-25September 1999, pp 213-25.

14

3.2 Conclusions

Having material to put into culture is the mainlimiting factor in oak somatic embryogenesis.Good acorn crops occur once every three to sevenyears and during the course of this project a goodacorn crop occurred only in 1996. In addition,there are no tested, proven superior families of oakthat would warrant the extra cost of vegetativepropagation.

Maintenance of oak embryogenic cell lines is nota problem. One cell line that has been in culturesince 1995 and has been maintained by subcultureevery six to eight weeks continues to producegood quality embryos after almost six years inculture. Similarly, formation of somatic embryosis also generally not a problem.

The main problem in oak somatic embryogenesisis turning off the embryo 'budding' programmeand turning on the embryo development andmaturation programme. This has also been aproblem in other plant species where secondarysomatic embryogenesis occurs. The highhumidity desiccation treatment and the high agartreatment show promise in helping to overcomethis bottleneck. Once mature somatic embryoscan be produced, they can be germinated andtransferred to the greenhouse where they appear togrow normally.

Finally, there is the question about the publicacceptance of ‘clonal oak’. Nevertheless, thistechnique could be a way to accelerate thepropagation of superior oak phenotypes once theyhave been identified in a tree improvementprogramme.

17

1. INTRODUCTION

Ash and sycamore are very important broadleavedspecies in Ireland. There has been little workundertaken so far on the genetic improvement ofash and sycamore. For many species, the use ofseed from the most productive provenances canyield a 4-6% increase in genetic gain/volume overunselected material. The selection of the bestperforming individuals can give a further gain of5-15%.

The basis for genetic improvement is to selectmaterial with a broad genetic base. Selected treesshould also be near maturity since their growthand morphological performance will be knownand also because the performance of juvenile treesis very difficult to predict with accuracy. Incollaboration with Coillte, the most productiveindividual trees with a superior stem form atmaturity were identified (selected ‘elite’ trees) inCoillte forests. Scions from the selected treeswere grafted. The grafted plants were conservedto form a core that would be a foundation forfurther genetic improvement. The material couldbe used in two ways: to produce seed progeny orto produce plants by vegetative propagation. Thevegetative propagation of selected trees allows theproduction of lines or varieties that are the exactgenetic copy of the original tree and offer greatpotential to increase the productivity and value ofnew plantations once they have been field tested.

Cuttings collected from mature trees generallyhave a low rooting percentage. This is due tophysiological changes that take place as the treegets older. Micropropagation and grafting arepotential methods to achieve vegetativepropagation from mature selected trees.Alternative approaches are either to rejuvenate theselected plants or to use material from donorplants, which may exhibit some juvenilecharacteristics (one such characteristic is thecapacity for rooting).

This project describes experiments aimed atestablishing methods for the vegetativepropagation of ash and sycamore usingconventional cuttings and micropropagation ofshoot cultures so that selected lines could beproduced on a large scale. The rooting capacitywas evaluated in cuttings taken from differentsources of donor plants: mature trees, graftedplants, re-grafted plants, seedlings, hedges, selfrooted cuttings and micropropagated plants.

2. MATERIALS AND METHODS

Approximately 100 elite ash and sycamore treeswere selected. The trees were defined as “clean,with straight stems and butts, free from knots, asround as possible with the heart straight down themiddle, free from wandering heart, with minimaltaper and without star or ring shake or epicormicburrs”.

Cuttings were collected from different types ofdonor plants as indicated. They were dipped inrooting powder (Seradix 3), except whereindicated otherwise, and inserted into a substrateof 2⁄3 peat and 1⁄3 perlite in 8 x 5 celled Hasseytrays. These were placed in an enclosed mist unitwith a mist burst of 18 seconds duration every 30minutes from 7 am to 8 pm daily. Rootingevaluations were made in October of each year.

For initiating shoot cultures, buds were sterilisedby placement in 0.1% w/v mercuric chloride for15 minutes, followed by one wash in sterile water,then a shake for 20 minutes in 7% w/v calciumhypochlorite and three rinses in sterile water.Single apical buds and nodes were cultured, oneper petri dish (5.0 cm diameter, 20 mm deep).Buds were transferred to fresh medium every 4-5weeks. When shoot elongation occurred, theshoots were cut in half and re-cultured. Later,explants were cultured consisting of: (i) a singleapical bud, (ii) single nodes and (iii), multi-nodalexplants consisting of at least two nodes, usuallyfrom the shoot bases which had 2-3 buds on shortinternodes. The basal medium (MS) for shootculture was Murashige and Skoog (1962) with B5Vitamins (Gamborg et al. 1968) instead of MSvitamins. The standard medium for ash shootculturing (M9) contained thidiazuron (TDZ), 1.1mg L-1, indole butyric acid (IBA), 0.2 mg L-1,benzyladenine (BA), 5.0 mg L-1 and sucrose 3%w/v. All media were agar solidified (Difco Bacto)8.5 g L-1, and pH adjusted to 5.8 beforeautoclaving. Shoots were initially cultured in petriplates and later in 150 ml glass powder jars withaluminium screw top lids with 35 ml ofmedium/jar and were transferred to fresh mediumevery 28 - 32 days. The light regime was 40 µEm2

sec-1 with a 16 hr-photoperiod and a temperature of22 ˚C ± 2 ˚C.

16

VEGETATIVE PROPAGATION OF SELECTED REPRODUCTIVESTOCKS OF ASH AND SYCAMORE

GERRY DOUGLAS1

SUMMARY

The feasibility of using vegetative propagation to provide plants from selected trees of ash (Fraxinusexcelsior L.) and sycamore (Acer pseudoplatanus L.) was determined. Grafting was the first method usedfor propagating plants from the mature selected trees. All ash clones and 73% of sycamore clones weresuccessfully grafted. Grafted plants were conserved in the nursery and field for further propagation.

Cuttings from grafted plants of sycamore gave 25% rooting. Cuttings taken from sycamore plants whichhad self rooted gave 49% rooting. This indicated that sycamore may have been rejuvenated and furtherimprovements in rooting rates may be possible.

Micropropagation of sycamore was difficult and gave low propagation rates; cutting propagation rather thanmicropropagation is a more practical option for sycamore. Although ash gave poor rooting rates in cuttings,micropropagation of several selected clones was successful. The culturing of ash buds with highconcentrations of cytokinins was necessary to establish viable cultures, but the regular transfer of culturesto cytokinin-free medium was necessary to maintain a healthy status in the cultures. In this protocol,spontaneous rooting in shoot cultures was observed and plants were successfully weaned to the glasshouse.

Established clones of ash were bulked up and are now available together with the method for large-scalevegetative propagation of selected lines of ash to provide plants for field testing.

1Teagasc, Kinsealy Research Centre, Horticulture and Farm Forestry, Malahide Road, Kinsealy, Dublin 17,

(gdouglas@kinsealy.teagasc.ie).

19

The rooting capacity of two types of cutting wascompared: apical 2-node cuttings with sub-apicaland 2-node cuttings with different auxin treatments.

Without auxin, rooting was low at 34 - 44% (Table3). The apical bud stimulated rooting but highrooting rates were also found in sub-apical two-nodecuttings (75%). Seradix rooting powder (8000 ppmIBA in talc) was superior to the proprietary product‘Hormone Rooting Powder’ (NAA 4000 ppm and2% captan).

A subsequent experiment confirmed that highrooting rates could be achieved with sub-apicalcuttings. Rooting in different substrates gave

different rooting rates: • 2⁄3 peat + 1⁄3 perlite gave 94% rooting in both apicaland subapical cuttings;• 2⁄3 peat and 1⁄3 peat nuggets (0-3 mm) gave 63% and94% rooting in apical and subapical cuttingsrespectively; • rooting was depressed with 2⁄3 peat and 1⁄3 peatnuggets (3-6 mm) and gave 55% and 75% rootingfor apical and subapical cuttings respectively.

These tests demonstrated that high rootingfrequencies could be achieved with ash cuttingssupplied with auxin. Furthermore, it is possible totake at least two cuttings from one shoot i.e. anapical and a subapical cutting.

FIGURE 1: ROOTING IN SEEDLING CUTTINGS OF ASH. Auxin (2000 ppm) applied to the cutting apex as in Table 2. Top: NAA treatment, bottom: IBA treatment.

Cutting type

Apex + 1st nodeApex + 1st nodeApex + 1st nodeSub-apical 2nd nodeSub-apical 2nd nodeSub-apical 2nd nodeApex + 2 nodesApex + 2 nodes

Auxin1

NoneSeradixHRPNoneSeradixHRPSeradixHRP

Rooting%4490753475688872

1Auxin treatments applied to the cutting bases. Seradix: IBA 8000 ppm in talc, HRP: NAA 4000 ppm + 2% captan inpowder.

TABLE 3: EFFECT OF SHOOT APEX AND AUXIN TREATMENT ON ROOTING CAPACITY OF ASHCUTTINGS DERIVED FROM SEEDLINGS (16-30 CUTTINGS/TREATMENT).

18

3. RESULTS AND DISCUSSION

Following identification of elite trees, scions werecollected and grafted onto 2-year-old rootstocksduring the dormant period using a cleft graft. All70 elite clones of ash gave viable grafts whereaswith sycamore 73% of clones gave viable grafts(Table 1). Scions collected from the successfulgrafts gave an improvement in the graft viabilitywith sycamore when grafts were made either inFebruary or July (Table 1). The grafted elite treesare now field planted at Coillte’s KilmacurraghNursery and at Kinsealy. In the Coillte nurserythey are widely spaced for seed production. AtKinsealy they are planted closer together to formhedges from which cuttings can be taken and thematerial propagated and used in clonal field testsin farm/forestry.

There is little previous research on rooting cuttingsfrom either juvenile or mature trees of ash. In thisstudy, juvenile cuttings were tested to define someparameters affecting rooting. It is necessary toknow the optimal conditions to achieve rooting in

cuttings, so that the method can be applied on alarger scale to cuttings from selected elite lines.Cuttings from different sources of donor plantswere used which may represent different states ofphysiological maturation corresponding tojuvenile, mature and rejuvenated states.

3.1 Rooting in cuttings derived from seedlingsand micropropagated plants

Table 2 summarises the rooting response of ashand the quality of roots formed with juvenilecuttings, which consisted of an apex plus twonodes. Auxin was mixed with lanolin as a pasteand applied to the apical buds of the cuttings.Application of auxin appeared to stimulaterooting. This suggests that the applied auxin wascapable of being absorbed at the apex and ofstimulating rooting at the base of the cutting.Without auxin treatment, 65% of the cuttingsrooted whereas 92% rooting was achieved with8000 mg L-1 IBA. In this case, 90% of the rootedcuttings had a strong rootball. The appearance ofroots is shown in Figure 1.

Ash

Sycamore

Origin of scions

mature treegrafted treemature treegrafted treegrafted tree

Grafting date

FebJuly1

FebFebJuly1

2Mean of cloneviability

%100 (70)100 (20)73 (70)

100 (20)73 (16)

Mean of graftviability

%9785255845

1Scions grafted by ‘tube’ method (Douglas et al. 1996). 2Numbers in parenthesis indicate the number of clones tested.

TABLE 1: VIABILITY OF GRAFTS OF ELITE ASH AND SYCAMORE WITH DIFFERENT SOURCES OFSCIONS AT DIFFERENT TIME PERIODS.

TABLE 2: EFFECT OF AUXIN TYPE AND CONCENTRATION ON ROOTING IN JUVENILE CUTTINGS OFASH.

Auxin1

treatment (mg L-1)NoneIBA (300)IBA (1000)IBA (2000)IBA (4000)IBA (8000)NAA (2000)

No. ofcuttings

55556055584950

013141818291417

11001004

251154614

336353631234433

Totalrooting

%65846865509282

Rooting class2

1Cuttings with apex plus two nodes, auxin applied to the cutting apical bud.IBA: indolebutyric acid, NAA: naphthalene acetic acid.

2Rooting class: 0: no roots, 1: roots 0.5 - 2.0 cm long, 2: roots 0.5 - 2.0 cm long and at least one root over 5 cm, 3: roots 0.5 - 2.0 cm long and two or more roots over 5 cm.

Cuttings were collected at the end of August andauxin was applied as commercial product or invarying amounts in lanolin.

Results from ash (Table 4) showed less rooting incoppice shoots compared to seedlings. However,an increased rooting rate with increasingconcentration of the auxin, IBA applied to the baseof the cutting (maximum 46%) was observed.Roots on plants derived from hedging werecoarser than those on cuttings taken fromseedlings (Figure 2). In the subsequent year, the influence of the stageof development of ash and sycamore shoots ontheir capacity for rooting in material from coppicewas investigated (Figure 3). Cuttings of ashcollected at the end of July gave a 10-fold increasein rooting percentage over those collected in mid-June. The later date of collection also yieldedbetter rooting in sycamore (Figure 3).

3.3 Cuttings from grafted elite clones of ash

In the first experiments, cuttings were taken fromgrafted plants of eight elite clones of ash, dippedin Seradix and placed in enclosed mist. Only oneclone rooted out of eight, with a rooting rate of8%. Poor rooting in cuttings from mature trees isoften attributed to physiological ageing and sometreatments such as re-grafting new shoots may

induce rejuvenation. In efforts to rejuvenate ash,scions from the original grafted plus trees were re-grafted on one, two, three or more successiveoccasions. The rooting capacity of cuttings,derived from plants that were grafted once, twiceand four times, was recorded for six elite clones.Rooting capacity was generally low in all material(5 - 20%) with 2/6 clones failing to root. None ofthe clones rooted in cuttings taken from plantswhich had been re-grafted four times. In addition,cuttings from plants of the clone Jenkinstown 47,which had been re-grafted three and four times,and from the clone Athenry 4, which had been re-grafted four, five and six times, were tested.Cuttings from plants of each re-grafted stage ofboth of these clones failed to produce roots. Theseobservations suggest that re-grafting had no effecton rooting capacity in cuttings.

3.4 Cuttings from grafted and self rooted eliteclones of sycamore

A comparison was made between the rootingcapacity of several sycamore clones using cuttingsderived either from grafted plants or from plantswhich were self rooted. A mean rooting rate of24% was obtained in cuttings from grafted plants.One clone failed to root out of eight tested (Table5). In contrast, a mean rooting rate of 49% wasobtained in cuttings from self-rooted clones and all

21

FIGURE 3 : EFFECT OF DATE OF COLLECTION ON ROOTING IN CUTTINGS FROM HEDGES OF ASHAND SYCAMORE. 86-121 cuttings/treatment, hedge flail cut in November annually, 15 different genotypes of each species.

Ash cuttings derived from apical cuttings and sub-apical cuttings were grown in 2l pots throughoutthe following season. Their height was measuredat the end of the growing season. The mean heightfor plants derived from cuttings with a shoot apexwas 79.38 cm ± 4.40 cm and for cuttings derivedfrom sub-apical cuttings was 78.65 cm ± 6.56 cm.The differences were not statistically significantshowing that there was no reduction in subsequentgrowth rates in cuttings which did not have a shootapex.

The rooting capacity in terminal shoot cuttingsderived from three clones of micropropagatedplants of seedling ash was also tested. At the time

of taking cuttings, the plants were pot bound.Rooting rate was clone dependant: giving 9% and26% for each of two clones and 77% for clone130/6 which also gave 81% rooting in cuttingsfrom lateral shoots.

3.2 Rooting of ash and sycamore cuttings fromcoppiced hedges

Large-scale production of cuttings would be fromhedges of elite clones. Annual trimming of thehedges would maintain them in a juvenile state.Rooting capacity was determined in cuttings froma hedge which was flail cut annually.

20

TABLE 4: EFFECT OF AUXIN TYPE, CONCENTRATION AND AUXIN CARRIER WHEN APPLIED TO THEBASE OF CUTTINGS DERIVED FROM COPPICED ASH (25-35 CUTTINGS/TREATMENT).

Auxin treatment (mg L-1)NoneIBA (1000)IBA (2000)IBA (4000)IBA (8000)IBA (8000)IBA (2000)NAA (4000)

Auxin carrier

LanolinLanolinLanolinLanolinLanolinTalc (Seradix)LanolinPowder (HRP)

Rooting%3030344043304637

FIGURE 2: ROOT FORMATION IN ASH CUTTINGS Roots emerging from the base of a Hassey tray after 12 weeks. Note more fibrous roots on cuttings taken from seedlings(left) and coarser roots on cuttings from hedged plants (right).

Cultures from clone Thomastown 70 weresuccessfully established with buds collected inMay 1998, from field-grown plants grafted once.In contrast, there was a failure to establish viablecultures from this same clone with buds collectedat the same time in May 1998, from glasshousegrown plants, which had been re-grafted threetimes. Similarly, with clones Jenkinstown 48 andAthenry 7; viable cultures were not establishedusing buds from plants which had been re-grafted3-5 times. These observations suggest that re-grafting (onto 2-year-old rootstocks) did notfacilitate the establishment of viable cultures fromseveral ash clones. Similarly, rooting was notimproved in cuttings from regrafted plants. It maybe concluded that re-grafting as performed here,was not effective in rejuvenating trees. Perhapsusing more juvenile rootstocks such as 1-year-oldrootstocks (rather than conventional 2-year-old),and larger pots, to give a greater development ofroots, may be a more effective method torejuvenate ash.

Care was taken not to sub-divide the shoot culturesexcessively during the first five subcultures sincethis caused necrosis of tissues and death of theshoot explants. Subculturing consisted ofbisecting any elongated shoots resulting in twotypes of explant, one with an apical bud whichcontinued to grow slowly and the other with

axillary buds, only one of which resumed growth.For both types of explant, the stem became highlylignified. In the first few subculture periods, thepropagation rate was less than one for most clones.The highest propagation rates were in the August -September period. Clone Monasterevin 72 wasthe most productive giving a maximum of fourexplants per original explant cultured.

The propagation rates achieved in these first trialswere too low for large-scale production of selectedlines so an alternative method for sub-dividing theshoots in established cultures was tested. Shootswere dissected to give three types of explant forculturing:(i) apical buds;(ii) individual nodes, consisting of two opposite

buds (with leaves detached); and (iii) multi nodal stems with basal callus attached.

In this way, a normal shoot could be sub-dividedinto three or four explants. The capacity of singlenodes to produce shoots from axillary buds issummarised for three clones in Table 9.

After 4 weeks of culturing, 71-92% of nodesproduced a shoot, depending on the clone andmedium used. Clone 8x was most responsive with70-96% of nodes producing a shoot. The higherconcentration of the cytokinin (TDZ), in medium

23

ChemicalNH

4NO

3

Ca(NO3)

24H

2O

K2SO

4

CaCl22H

2O

Solution 41

H3BO

3

Na2MoO

42H

2O

MgSO47H

2O

MnSO47H

20

ZnSO47H

2O

CuSO45H

2O

Thiamine HClInositolSequestrene Fe 3302

Final concentration mg L-1

400.00

556.00

990.00

96.00

10.00(ml)

6.20

0.25

370.00

22.30

8.60

0.25

0.25

50.00

40.00

TABLE 6: COMPOSITION OF QRC MEDIUM.

1 Solution 4 i.e. Ammonium phosphate instead of potassium phosphatePreparation Steps for Solution 4a. 0.053g (NH

4)

2HPO

4in 50 ml H

2O

b. 0.573 g NH4H

2PO

4 in 50 ml H

2O = (0.1 M)

c. Take 45 ml solution No. 2 and add to 50 ml solution No. 1d. Adjust pH to 5.8 using No. 2 solution = Solution 4e. Bring to final volume 100 mlf. Store in freezerg. Use 10 ml of Solution 4 per 1.0 L medium2 Sequestrene Fe 330 (Geigy) instead of Fe-EDTA

clones rooted. These observations suggest thatself-rooted mother plants can be a source ofcuttings with an improved capacity for rooting.Lower rooting rates in cuttings from grafted plantsmay be due to a greater physiological ageing ingrafted plants or graft/scion incompatibilities.

3.5 Micropropagation of sycamore and ash

Sycamore clones were established in vitro usingbuds from grafted elite clones. Buds werecultured on QRC basal medium without growthregulators, containing 3 g L-1 charcoal (Table 6).High levels of bud sterility were obtained; resultsare summarised in Figure 4. Sycamore culturesfailed to respond to applied cytokinins as otherspecies. The establishment of an efficientmicropropagation system was not possible.However, since sycamore can be propagatedsuccessfully by cuttings, it is proposed to usecuttings rather than micropropagated plants as thepreferred method of providing plants for fieldtesting.

There is little practical experience of clonal propagation of Fraxinus species. Micro-propagation protocols have been published forjuvenile material (Hammatt and Ridout 1992, Kimet al. 1997). For mature ash, a single clone wasestablished in micropropagation by Hammatt(1994) and another by Pierik (1999). In this study,the hormone regime of Kim et al. (1997) was usedto initiate and establish viable cultures from arange of ash clones.

Buds were collected from 40 selected trees inspring when bud flushing had just started (Table7). Some trees provided over 30 buds forinitiation of shoot cultures, others were small andless than 10 buds per clone were available.Surface sterilisation was satisfactory; 90% of theclones were free from surface contamination after4 weeks. Out of 845 buds sterilised, 522 (62%)were free of fungal or bacterial contamination.

The viability of sterile buds from each of the 40clones was tested over successive periods ofculturing (Table 8). Buds were cultured on M9medium. At the end of the second culture period,40% of clones were necrotic, leaving 60% stillviable. Thereafter, most clones survived for twofurther culture periods but by the sixth cultureperiod only 20% of clones remained viable. Allfive clones which survived to the seventh cultureperiod (12.5% of original amount) remainedviable thereafter and became established as shoot-producing cultures.

The high mortality rate of ash buds during theestablishment phase of shoot cultures has beenattributed to the physiological maturity of thedonor tissues. Successive grafting of new growthfrom grafted plants has been reported to inducerejuvenation in the grafted scion material. Overseveral years a re-grafting programme was carriedout with selected clones of ash. The capacity ofbuds from some re-grafted clones to becomeestablished in vitro as viable cultures wasobserved.

22

Clone

Gorey 60Piltown 34Bram 220-8Bram 223-4Piltown 40Camolin 81Durrow 79Durrow 75Athenry 1Athenry 2Athenry 6Knocktopher 45Durrow 66Mean rooting

Cuttings from grafted plants

n.t.1

n.t.41.6n.t.

11.1n.t.n.t.

27.030.021.425.036.60.0

24%

Cuttings from self-rooted plants

81.875.073.352.630.016.613.3n.t.n.t.n.t.n.t.n.t.n.t.

49%

TABLE 5: ROOTING CAPACITY IN CUTTINGS DERIVED FROM GRAFTED PLANTS OF ELITE CLONES OFSYCAMORE AND IN CUTTINGS COLLECTED FROM CUTTING-DERIVED PLANTS (SELF ROOTED).

Rooting/clone%

1n.t. = not tested

shoot cultures were transferred to hormone-freemedium (QRC) containing 3.0 g L-1 activatedcharcoal. On this medium, the explants reverted toproduce healthy shoots, which could be furtherpropagated. The yields of explants obtained onQRC and M9, from three types of original explant(apex, single node and multiple node) for twoclones are summarised. Although the propagationrate declined by culturing explants without growthregulators (Table 10), the new shoots producedwere healthy in appearance and often rootedspontaneously. Spontaneous rooting may be

indicative of rejuvenation. To maintain the healthof cultures and consistent rates of propagation thematerial was routinely cultured onto mediacontaining growth regulators (M9) followed by aperiod without growth regulators (QRC).

The rooting capacity of six seedling clones in vitroand the effects of three concentrations of IBA wastested (Table 11). One-hundred percent rootingwas achieved for three clones; the optimalconcentration of IBA varied for each clone. Forthe mature clone 8x, the initial rooting trial gave

25

Medium1

M9N9N13N14

F5% mm82 2.729 1.139 1.350 3.2

JK47% mm71 5.771 2.957 1.7

-

8x% mm92 2.396 8.896 10.270 5.8

% of nodes producing shoots in clones (mean shoot length)

TABLE 9: EFFECT OF FOUR MEDIA ON PRODUCTION OF SHOOTS FROM SINGLE NODE EXPLANTS OFTHREE MATURE CLONES OF ASH IN VITRO.

TABLE 10: EFFECT OF MEDIA ON PRODUCTION OF EXPLANTS BY APICES, SINGLE NODES ANDMULTI-NODES IN TWO ELITE CLONES OF ASH CULTURES.

1M9: TDZ 1.1 mg L-1, BA, 5.0 mg L-1

N9: TDZ 0.11 mg L-1, PBA, 2.5 mg L-1

N13: TDZ 0.11 mg L-1

N14: TDZ 1.1 mg L-1, PBA, 2.5 mg L-1

Clone

Monasterevin 72

Jenkinstown 49

Medium1

QRCM9QRCM9

1.23.21.01.0

1.23.91.0

-

2.62.62.71.3

Mean yield of explants obtained per:shoot apex single node multi node

1M9: MS medium + TDZ 1.1 mg L-1, BA 5.0 mg L-1, IBA 0.2 mg L-1. QRC: Basal medium, no hormones, 3% charcoal (Table 10).

TABLE 8: INITIATION OF CULTURES OF 40 SELECTED MATURE ASH IN 1998, VIABILITY OF BUDS ATTHE END OF EACH SUCCESSIVE PERIOD OF CULTURING.

Cultureperiod1st

2nd

3rd

4th

5th

6th

7th 1

No. clones viable

36241515985

Clones viable %

90.060.037.537.522.520.012.5

Clones lost%

10.030.022.50.0

15.02.57.5

Number of viable clonesat each culture period

40292020995

1Clones which were viable after seven subcultures remained viable and became established as shoot producing cultures. M9medium used throughout i.e. MS medium + TDZ 1.1 mg L-1, BA 5.0 mg L-1, IBA 0.2 mg L-1. The five viable clones wereAthenry 8 and 6, Thomastown 70, Monasterevin 72 and Shelton 10.

M9, favoured shoot development from singlenodes. Nodes of this clone which were reculturedtwice, without dissection, to fresh media resultedin final shoot lengths of 22, 25, 23 and 19 mm onmedia M9, N9, N13, and N14 respectively. Inaddition, these cultures also produced secondaryshoots in 47% of cultures on M9, 17% on N9, 31%on N13 and 6% on N14. From this and otherexperiments, the hormone regime of M9 wasadopted as the standard medium and its effectstested on shoot production in four juvenile

seedling clones of ash. Nodes and apices wereused as the explants and micropropagation rates of4.1, 2.4, 5.2, and 3.4 were obtained for eachgenotype respectively.

The appearance of ash shoots which were culturedfor several months on the standard medium M9became glassy, water-soaked and unhealthy(vitrification). This abnormality is generallycaused by excessive cytokinins in the medium andcan lead to the loss of cultures. Therefore, the

24

FIGURE 4 : VIABILITY OF SELECTED CLONES OF SYCAMORE AFTER THREE SUBCULTURES ON QRCMEDIUM

Forest

AthenryAthenryAthenryAthenryAthenryAthyAthyAthyAthyAvonmoreCastlecomerCastlecomerCastlecomer

Designatednumber

678

38533637515282404146

Forest

CastlecomerCongDonadeaDundrumDundrumDurrowJenkinstownJenkinstownKnocktopherKnocktopherKnocktopherMonasterevinMonasterevin

Designatednumber

472

2380815448496475767273

Forest

MonasterevinMonasterevinPortlaoisePortlaoisePortlaoiseRoscreaSheltonSheltonSheltonShillelaghStradballyThomastownVirginia

Designatednumber

74777879851

10111262847083

1Each plus tree has been grafted.

TABLE 7: DESIGNATION OF 40 ASH PLUS TREES1 FROM WHICH BUDS WERE INITIATED INTO MICROPROPAGATION IN 1998.

%

4. CONCLUSIONS

1. Vegetative propagation of ash and sycamore is a feasible approach to use as one element in the genetic improvement of these important broadleaves.

2. Elite trees were selected among all mature trees in our native populations; grafting resulted in the conservation of all elite ash and 73% of elite sycamore.

3. Rooting rates of up to 90% were obtained for ash cuttings that were collected from juvenile seedlings, indicating a high rooting potential among many genotypes.

4. The propagation of mature, elite ash trees by cuttings is still not optimal using material from grafted plants. However, rooting of cuttings collected from micropropagated plants is more promising.

5. Propagation of elite ash trees by micropropagation was possible for 12.5% of the clones tested. Micropropagation allows for the large-scale production of plants and may provide rejuvenated plants with a high rooting capacity in cuttings from micropropagated donor plants.

6. The efficiency in micropropagation of ash was improved by using single buds and nodes as the cultured explants rather than whole or bisected shoots.

7. We established shoot cultures from eleven elite sycamore clones. However, micropropagation of sycamore requires more development since the propagation rates obtained were too low for practical use.

8. Collecting cuttings of ash and sycamore from elite plants which are maintained as hedges may provide a practical way of maintaining elite plants which provide a succession of cuttings with a high capacity for rooting.

9. Serial grafting (re-grafting) of ash was not a successful method of rejuvenating plants.There was no increase in the capacity of cuttings to root or of buds to become established as viable shoot cultures, in material which was re-grafted many times.

10. Sycamore cuttings showed a high rooting capacity among 13 clones tested and 25% of cuttings from grafted plants produced roots. When cuttings were taken from these rooted plants the rooting percentage increased to49%. This indicates that some rejuvenation, and recovery of rooting capacity may have taken place in sycamore. This would offer the potential to develop large-scale vegetative propagation of sycamore via cuttings.

2726

IBA mg L-1

01.05.010.0

83-

50-

12-

33-

-91

100-

89100100100

77-

10085

-8969-

Seedling genotype 1 2 3 4 5 6

Rooting per clone %

TABLE 11: ROOTING IN SIX DIFFERENT GENOTYPES OF ASH AND THE EFFECTS OF INDOLE BUTYRICACID (IBA) MS MEDIUM 1

10 STRENGTH, SUCROSE 3%.

58% rooting with 5.0 mg L-1 IBA and 40% in theabsence of IBA. Root number per cutting was 2.3with IBA and 1.3 in the absence of IBA. Thesestudies indicated that rooting was stimulated byIBA and rooting potential was high in seedling aswell as in micropropagated mature clones of ash.

Using the micropropagation methods describedabove for the selected ash clones from maturetrees, approximately 100 plants were producedwhich were transferred to the greenhouse forfurther field testing (Figure 5).

FIGURE 5: MICROPROPAGATED PLANTS OF SELECTED MATURE ASH TREES IN THE GREENHOUSEAT THE END OF THE FIRST GROWING SEASON.

PHOTOAUTOTROPHIC MICROPROPAGATION OF ASH ANDSYCAMORE

MICHAEL J. HENNERTY1, NASRIN NAKHSHAB2 AND ROGER LONG3

SUMMARY

Ash (Fraxinus excelsior L.) and sycamore (Acer pseudoplatanus L.) are important forest tree species inIreland. Methods of vegetative propagation should be available if the genetic potential available ofindigenous elite populations is to be exploited in the short to medium term.

Two methods of vegetative propagation of these species have been studied previously, production byconventional cuttings and by conventional micropropagation. Use of conventional cuttings is possible inthe case of sycamore but it is not an efficient system and the economic costs are deemed too high for thelevel of gain likely to be achieved. In the case of ash it is simply not possible to root conventional cuttingsat anything other than very low, uneconomic rates. Micropropagation has long been suggested as a potentialalternative to traditional methods of vegetative propagation. Unfortunately, in spite of many years ofresearch effort around the globe, this approach has not been successful in developing a consistentmethodology at the laboratory level, much less a fully commercial system.

In the course of this study, much of the published work on conventional micropropagation of these specieswas repeated for comparison purposes and many of the problems experienced by other researchers wereconfirmed. Photoautotrophic micropropagation methods were investigated as a possible way of overcomingthe major hurdles experienced with conventional micropropagation. Procedures were developed for thedirect establishment of material from mature sycamore under photoautotrophic conditions. This was bestachieved using semi-hardwood explants in the summer and autumn months. This material was then used ina microcoppice or microstoolbed to produce a supply of softwood microcuttings for rooting, weaning andestablishment. In the case of ash, direct establishment of microstoolbeds was not possible but bud break andstem elongation from material excised from mature trees was achieved under photoautotrophic conditionsat any time of the year. The shoots from these cultures were then used to establish microstoolbeds. Withsuccessive harvesting of microcuttings from these cultures the percentage of successful rooting increased to90%, offering for the first time a potentially commercial method for the micropropagation of ash selectedfrom mature trees. In the case of both species, it has now been shown that there is a pathway for efficientvegetative propagation suitable for transfer to the industry.

The level of regeneration, the numbers of microcuttings obtained from the microstoolbeds, the longevity ofthe cultures, the possible manipulation by plant growth regulators and other factors such as the size, numberand quality of the cuttings all require further work for the full potential of the technology to be elucidatedand before a full economic analysis of the system can be concluded.

29

1. Michael J. Hennerty, Dept. of Crop Science, Horticulture and Forestry, Faculty of Agriculture, UCD, Belfield, Dublin 4 (michael.hennerty@ucd.ie).

2. Nasrin Nakhshab, Bord na Mona, Main street, Newbridge, Co Kildare, (nasrin.nakhshab@bnm.ie).3. Roger Long, Green Crop Ltd., Carlow, Co Carlow.

28

5. REFERENCES

Douglas, G. C., McNamara, J. and Thompson, D.1996. A tube method for grafting small diameterscions of the hardwoods Quercus, Fraxinus,Betula and Sorbus in Summer. Int. Plant Prop.Soc. 46: 221-226.

Hammatt, N. 1994. Shoot initiation in the leafletaxils of compound leaves from micropropagatedshoots of juvenile and mature common ash(Fraxinus excelsior L.). Journal of ExperimentalBotany 45 (275): 871-875.

Hammatt, N. 1996. Fraxinus excelsior L.(Common Ash). In: Biotechnology in Agricultureand Forestry 35, Trees IV, Ed. Bajaj, Y.P.S.Springer-Verlag, Berlin, Heidelberg, New York.

Hammatt, N. and Ridout, M. 1992.Micropropagation of common ash (Fraxinusexcelsior). Plant Cell, Tissue and Organ Culture15: 67-74.

Kim, M. S., Schumann, C. M. and Klopfenstein,N. B. 1997. Effects of thidiazuron andbenzyladenine on axillary shoot proliferation ofthree green ash (Fraxinus pennsylvanica Marsh.)clones. Plant Cell Tissue and Organ Culture 48:45-52.

Gamborg, O. L., Miller R. A. and Ojima, K. 1968.Nutrient requirements of suspension cultures ofsoybean root cells. Exp. Cell. Res. 50: 151-158.

Murashige, T. and Skoog, F. 1962. A revisedmedium for rapid growth and bioassays withtobacco tissue culture. Physiol. Plant. 15: 473 -497.

Pierik, R. L. M. 1999. Personal communication.Wageningen Agricultural University, Dept. ofHorticulture, Wageningen, The Netherlands.

2. MATERIALS AND METHODS

Various source materials were used to establishcultures of both sycamore and ash. Theseincluded seeds, dormant buds from both juvenileand mature sources, shoot cuttings from bothjuvenile and mature sources and epicormic shoots.

2.1 Medium preparation and use

Throughout this study a variety of plant tissueculture media were used. These included MSmedium (Murashige and Skoog 1962), WoodyPlant medium (WPM) (Lloyd and McCown 1980),DKW medium (Driver and Kuniyuki 1984) andEnshi medium (Hori 1966). All of the media weremade up from analar grade reagents maintained asstock solutions in a cold room (4 ˚C) or freezer (-20 ˚C) and were autoclaved at 121 ˚C, 1.03 Nm-2 prior to use.

A variety of substrate were used. Agar (SigmaType IV) was used as the primary gelling agent.In addition gelrite, vermiculite, perlite and avariety of horticultural polyurethane foamsubstrates were used.

2.2 Growth room conditions

Cultures were placed in conventional growthrooms at a temperature of 20 ± 2 ˚C, with a 16 hr-photoperiod and a photosynthetic photon fluxdensity (PPFD) of 30 µmol s-1m-2. Forphotoautotrophic conditions the cultures wereplaced in a growth room with either 90 or 130µmol s-1m-2, with a 16 hr-photoperiod, carbondioxide enrichment (CDE) external to the culturevessel of 3000 ppm and a temperature of 23 ± 2 ˚C.

2.3 Establishment and culture of ash seed invitro

Fresh or dry seeds were immersed in 1% sodiumhypochlorite (w/v) with a few drops of 0.01%Tween for 20 for 30 minutes. They then wererinsed five times with sterile distilled water. Theapical third of seeds (opposite radicle end) wasaseptically excised prior to inoculation in testtubes containing shoot induction medium (halfstrength MS + 3% sucrose, 0.7% agar, pH 5.7).

2.4 Establishment and culture of dry seed ofsycamore in vitro

Sycamore seeds (samaras) were collected from asingle tree on the Belfield campus of UniversityCollege Dublin in October 1995. Dry seeds werepicked from the ground and stored until used.Samaras were washed in running tap water andsoaked in 5% Domestos overnight. After rinsingseveral times in distilled water, the pericarp wasremoved and the seeds were soaked in 1.0 g L-1

Benlate for 30 minutes and rinsed thoroughly withdistilled water. Seeds were transferred to 20%calcium hypochlorite solution for 30 minutes andwashed 3 times in sterile distilled water. Seedswere then placed in sterile petri dishes onautoclaved filter paper moistened with steriledistilled water and incubated for 48 hrs at 21 ± 3 ˚C in the dark. The testa of each seed wasthen removed and each was sterilised byimmersion in 85% ethanol for 30 seconds,followed by 15 minutes in 7% calciumhypochlorite. Seeds were rinsed five times insterile distilled water and cultured in half strengthMS medium with 6.0 g L-1 agar, 3% sucrose, andpH 5.6 - 5.8.

2.5 Establishment and culture of fresh seed ofsycamore and ash in vitro under enrichedcarbon dioxide conditions

Fresh seeds of ash and sycamore were collected inthe middle of September 1997. Samaras werewashed for one hour under running tap water andsterilised with 5% Domestos for 30 minutes.Seeds were extracted from the samaras and theirpericarps were removed. The testa of eachsycamore seed was removed and the seeds weresoaked in 0.1% Captan for 30 minutes, thentransferred to 7% calcium hypochlorite for 15minutes. After washing five times with steriledistilled water, sycamore seeds were cultured intest tubes containing half strength MS sugar freemedium with 0.6% agar, pH 5.7.

With ash it was necessary to aseptically excise onethird of each seed opposite the radicular end priorto inoculation in order to facilitate gas diffusionand cotyledon emergence. The seeds werecultured in test tubes containing half strengthsugar-free MS medium with 0.6% agar, pH 5.7.All cultures were held at 20-24 ˚C with a 16 hr-photoperiod and a light intensity of 130 µmol s-1m-2.

3130

1. INTRODUCTION

The broadleaved species of interest to Irishforestry are all outbreeders. The ability to clonallypropagate them in order to multiply individuals orfull-sib families from breeding programmes thatexhibit advantageous characteristics may offerstrategic advantages to the industry.

The usual approach to clonal propagation is byeither in vivo cuttings or conventionalmicropropagation. The relative merits of thesemethods are well documented. Propagation byrooted cuttings is slow, seasonal and at bestexhibits variable success rates. Conventionalmicropropagation is a technique that is applied tohigh value ornamental and plantation crops. Theproblems and costs associated with conventionalmicropropagation are such that it is not now, nor isit ever likely to be, a cost effective technique forthe production of large volumes of forestryplanting material, unless a large commercialadvantage is to be gained from the use of eliteplanting material.

There are two alternatives to conventionalmicropropagation that offer a potential solution tosome of the problems associated with thattechnique. These are somatic embryogenesis andphotoautotrophic micropropagation. Somaticembryogenesis is considered by many authoritiesto be the long-term future for clonal propagationof forestry species.

Photoautotrophic micropropagation is a method ofmicropropagation where the sucrose is removedfrom the tissue culture medium and the plantmaterial is placed in conditions of enhanced lightlevel and elevated carbon dioxide concentrationsto encourage the material to photosynthesise. Thepresence of sucrose, leading to a highcontamination risk, physiological problems andthe use of small containers is now recognised as amajor factor associated with production problemsand high production costs in conventionalmicropropagation.

The objective of this project was to examine thetechnique of photoautotrophic micropropagationand its application to ash and sycamore anddevelop a cost effective technique that would be ofapplication in Irish forestry, either in theproduction of clonally propagated material forplantation establishment or as a tool in thedevelopment of the wider scale use of adaptedgermplasm in Ireland.

Hammatt (1996) reported that an immediate andkey objective of ash biotechnology is to developmeans to clonally propagate selected trees, thusenabling rapid genetic gain to be achieved withthis species, which has been studied littlegenetically, and has a long breeding cycle,flowering when 15-20 years old. Givendifficulties with the rooting of cuttings, successfulpropagation in vitro could be used to generatedirect planting stock for both clonal trials andcommercial woodland.

3. EXPERIMENTAL WORK: RESULTSAND DISCUSSION

3.1 Establishment of in vitro cultures

A number of experiments were performed toestablish selected material in in vitro culture. Inan ideal situation, the ability to select materialfrom semi-mature or mature trees and multiply itin vitro would be desirable. The literatureindicates that this is practically impossible withoutfirst inducing a phase change back to juvenilecharacteristics. In order to ensure a supply ofmaterial for further experimentation, the approachtaken here was to use the traditional route ofinitiating cultures from germinating seeds undermixotrophic conditions. However, more novelapproaches such as germinating seeds underphotoautotrophic conditions, the use of juvenileand mature shoot cuttings containing dormantbuds, the use of softwood and semi-hardwoodcuttings as explants and finally the induction ofepicormic shoots from the trunks of mature treesfor use as explants were also attempted.

3.1.1 Seed germination under mixotrophicconditions

Ash and sycamore seeds were treated aspreviously described (Section 2.3 and 2.4) andplaced on 1/2 MS medium containing 3% sucrosesolidified with agar. These were placed in agrowth room under the standard mixotrophicconditions. In more than 95% of the cultures,fungal and bacterial contaminants quicklyoverwhelmed the cultures and the material waslost. This was in spite of repeated attempts withvarious seed lots of both fresh and aged seed atdifferent times of the year.

3.1.2 Seed germination under photoautotrophicconditions

Seeds were treated as described previously(Section 2.5) and placed on 1/2 MS sugar-freemedium solidified with agar and cultured underphotoautotrophic conditions. With sycamore, thisresulted in 100% germination and no visiblefungal contamination of the cultures. Bacterialcontaminants were visibly present inapproximately 5% of cultures. The seedsgerminated approximately one week aftercommencement of in vitro culture; 15-20 cmgrowth was recorded after four weeks.

Germination of ash seed was much lower, 22%,possibly as a result of seed dormancy due to theunder-development of the embryo at the time offruit collection. This is well documented in theliterature (Boner 1974, Young and Young 1992)and can be overcome by after-ripening the fruitand/or by stratification. Such techniques were notattempted within this study.

Culture of seeds on sugar-free medium under CDEconditions provides for fast in vitro seedlingestablishment with a high percentage of seedgermination (particularly in the case of sycamore)and virtually no visual contamination bysaprophytic organisms. This is a valuable optionto establish in vitro seedling cultures from elitematerials, particularly where the supply ofmaterial is limited and the risk of loss of materialthrough contaminating fungal and bacterialspecies is required to be minimised. Excellentgermination and initial growth was achieved underthese conditions and while there was little or noobvious contamination problems, the materialcould not be regarded as axenic.

3.1.3 Direct establishment of sycamore semi-hardwood cuttings under autotrophic conditions

A trial was set up examining the effect of differenthormone rooting treatments and substrates onrooting ability. The nutrient medium used wasEnshi. Hormone rooting treatments consisted ofplacing the basal end of the cutting into either acommercial rooting powder, or IBA solutions of2500, 5000, or 10000 ppm. The control consistedof a dip in distilled water. Thirty replicate cuttingswere used for each hormone/substratecombination.

The type of substrate, concentration of IBA andtheir interactions were all significant. Thehortifoam substrate yielded a significantly higherpercentage rooting than vermiculite, orvermiculite plus gelrite. Rooting powder was themost effective hormone treatment for rooting ofsycamore semi-hardwood cuttings (Table 1).

There was a significant interaction betweensubstrate and hormone. The maximum rootingoccurred in the cuttings, which were planted in hortifoam and treated with rooting powder(Table 2).

In a separate trial the inclusion of IBA in themedium (in contrast to a dip of the basal end of thecutting in IBA) at a range of concentrations, was

3332

2.6 Direct establishment of sycamore softwoodand semi-hardwood cuttings under enrichedcarbon dioxide conditions

Cuttings were collected from sycamore trees on theBelfield campus in July 1997. Cuttings werewashed for 30-60 minutes under running tap water,soaked for 30 minutes in 1.0 g L-1 Benlate,followed by surface sterilisation in 5% calciumhypochlorite with a few drops of Tween 20 andthen rinsed five times with sterile distilled water.Each cutting with two nodes was treated witheither rooting powders or a dip in an auxinsolution. Cuttings were cultured immediately insterilised Magenta vessels with vented GA-7 lidscontaining sugar and growth regulator (GR) freeliquid Enshi medium. Vermiculite, hortifoam andvermiculite plus gelrite were used as substrates.Cultures were transferred into CDE (3000 ppmCO

2) conditions at 21-24 ˚C with a 16 hr-

photoperiod and a light intensity of 90 µmol s-1m-2.

2.7 Production of epicormic shoots frommature ash and sycamore

Trees approximately 20 years old, growing on theBelfield campus, were felled and the trunks cutinto logs about 40 cm long. Logs from eachspecies were soaked for 2 hours in 1 mg L-1 GA

3,

BAP, or TDZ and left standing in 50 cm pots ofShamrock potting compost (Bord na Mona,Ireland) and covered with clear plastic in a heatedglasshouse. The number of epicormic shootsproduced was counted after two months.

2.8 Experimental designs and statisticalanalyses

The experimental designs used are outlined in theresults section but usually consisted either of acompletely randomised block design, or a factorialrandomised block design where two or moretreatments were being examined at a number ofdifferent levels. The data obtained were analysedusing the Statistical Analysis System using ageneral linear model (SAS 1985).

3.1.5 Disinfection procedures

One of the major problems in micropropagation ofwoody species is the introduction of material intoculture and removal of contaminating fungi andbacteria from the explants. This was reinforced bythe experiences during this project. While theabsence of sucrose in the medium meant thatcontaminating microorganisms did not havereadily utilisable substrate to grow on, it was stilldesirable and represents good laboratory practiceto disinfect the starting material. In this trial, theeffect of various disinfection procedures inremoving contaminating microorganisms fromsycamore semi-hardwood nodal explants wasexamined.

Sycamore semi-hardwood cuttings were takenfrom mature trees on the Belfield campus.Explants consisting of two nodes were treated asdescribed in Section 2.7. After the initial tap waterwash, material was either treated with Benlate orleft untreated. Explants were then subjected todisinfection using one of four disinfectants at fourdifferent concentrations. After further washing insterile distilled water the explants were placed onhalf strength MS medium containing sucrose (30 gL-1) and solidified with agar. The number ofcontaminated nodes was counted and the type ofcontaminant assessed; the number of damaged ornecrotic nodes and the number of nodes exhibitinga halo of dark phenolic substances in the agar wererecorded.

In all cases, where explants were pre-washed inBenlate, the level of contamination wassignificantly decreased. Domestos was lesseffective than other disinfectants in reducing thelevel of fungal contamination (Table 4). None ofthe disinfectant treatments used was effective

against bacterial contaminants and there were nosignificant differences in that regard. Thispresumably reflects the fact that the bacterialcontaminants were either endogenous in origin orwere protected in some manner on the epidermisof the explant during the disinfection procedure.Calcium hypochlorite caused significantly lessdamage on sycamore explants and there weresignificant differences between calciumhypochlorite and the other disinfectants. Surfacesterilisation with calcium hypochlorite resulted inless phenolic compounds being secreted in themedium as measured by visual assessment of thecultures (Table 4).

3.1.6 General discussion on the establishmentphase

Establishment of material from woody plants in invitro culture is always problematic. One of themajor barriers to success is the large exogenousand endogenous microflora that quickly over-runthe medium and destroy the culture. Anothermajor problem is the presence of phenoliccompounds that move out of the explant into themedium preventing or stunting growth and oftencontributing to the death of the explant. Trialsconducted in the course of this study haveconfirmed these problems, particularly whenmaterial is placed under mixotrophic conditions,with sucrose present in the medium.

Progress in tissue culture of ash has been madeonly relative recently, and all of the reports havebeen with juvenile material (Chalupa 1983 and1993, Hammatt and Ridout 1992, Hammatt 1994).Hammatt and Ridout (1992) used seedlingsbecause their early work with mature ash hadfailed due to contaminating microorganisms in the

35

Substrate

Hortifoam Hortifoam Hortifoam Hortifoam VermiculiteVermiculiteVermiculiteVermiculite

IBA mg L-1

0.000.250.501.000.000.250.501.00

Rooting (0 day)%13196

312

252

312

442

312

TABLE 3: EFFECT OF DIFFERENT CONCENTRATIONS OF IBA AND TYPE OF SUBSTRATE ON ROOTINGOF SYCAMORE SEMI-HARDWOOD CUTTINGS (WITHOUT COLD TREATMENT).

1p ≤ 0.05 2p ≤ 0.001 3p ≤ 0.0001

34

used in an attempt to root sycamore semi-hardwood cuttings. In addition, a cold pre-treatment (4 ˚C in the dark) was used in an attemptto improve the percentage of cuttings rooted. Inthis case, vermiculite was significantly superior tohortifoam in the control treatment. IBA at 1.0 mgL-l significantly increased the percentage rooting inthe case of hortifoam, but there was no significantincrease when vermiculite was used as a substrateand at this level of IBA there were no significantdifferences between the substrates (Table 3).

Cold pre-treatment for two days prior toinoculation did not significantly increase thepercentage of cuttings rooted. Longer pre-treatment led to higher levels of necrosis and deathof the cultures. Nodal cuttings, where theysurvived, produced callus at the basipetal end ofthe cuttings but the level of rooting was poor.

3.1.4 Production and culture of epicormic shootsfrom mature ash and sycamore

Epicormic shoots from both ash and sycamorewere produced, in the manner previouslydescribed (Section 2.7), from logs in September.For both species approximately five or six shootsup to 12 cm in length were produced within a two-month period, irrespective of hormone pre-treatment used. Attempts to introduce these shootsto culture resulted in a large percentage of thematerial becoming contaminated, primarily withalgal growth. This arose mainly due to the use ofnon-sterilised peat based compost for standing theshoots in and the use of polythene bags to maintainhumidity.

IBA concentration (ppm)02500500010000Rooting powder

Rooting%

18c1

11c11c27b47a

Substrate

HortifoamVermiculiteVermiculite + gelrite

Rooting %

41a5c

21b

1Means followed by the same letter are not significantly different, (p ≤ 0.05).

TABLE 1: EFFECT OF DIFFERENT CONCENTRATIONS OF IBA, ROOTING POWDER AND SUBSTRATETYPE ON ROOTING OF SYCAMORE SEMI-HARDWOOD CUTTINGS.

Substrate

Hortifoam Hortifoam Hortifoam Hortifoam Hortifoam VermiculiteVermiculiteVermiculiteVermiculiteVermiculiteVermiculite + gelriteVermiculite + gelriteVermiculite + gelriteVermiculite + gelriteVermiculite + gelrite

Hormone ppm

02500

5000010000

Rooting powder0

25005000

10000Rooting powder

025005000

10000Rooting powder

Rooting %403

333

201

272

873

00

190

1313473

07

403

TABLE 2: EFFECT OF SUBSTRATE TYPE AND PGR ON ROOTING OF SYCAMORE SEMI-HARDWOODCUTTINGS.

1p ≤ 0.05 2p ≤ 0.001 3p ≤ 0.0001

3.2.1 Effect of medium, BAP and activatedcharcoal on shoot growth and multiplication rateof ash and sycamore nodal explants

Shoots from in vitro grown seedlings of ash wereremoved from the tissue culture jars and dividedinto nodal segments, with two buds on eachsegment. Leaves subtending the buds wereremoved. These were then cultured on media inWilsanco tubs using WPM and DKW media, withthree different concentrations of BAP and in thepresence or absence of activated charcoal. Theexperiment consisted of a factorial arrangement ina completely randomised design.

Activated charcoal, concentration of BAP and theinteraction between BAP concentrations andactivated charcoal were significant for shootheight, multiplication rate and callus formation atthe base of nodal explants (Table 5). Theinteraction between media and activated charcoalwas significant on shoot height. DKW wassignificantly better than WPM for shoot height ofnodal explants. Minimum shoot multiplicationand callus formation was in a medium containing1.0 mg L-1 BAP which was significantly differentfrom the higher concentrations of 5.0 and 2.5 mg L-1 BAP. The shoot height of nodal explants wasmaximum in a medium with 2.5 mg L-1 BAP,median with 1.0 mg L-1 BAP and was least at 5.0mg L-1 BAP and differences between the threeconcentrations were significant (Table 5).

Medium without activated charcoal significantlyincreased shoot multiplication, shoot height andcallus formation at the base of cuttings (Table 6).

In general, DKW medium was better than WPMmedium for shoot growth and elongation while themultiplication rate was not significantly affectedby medium. Maximum shoot multiplication (2.71± 1.25) and shoot growth (3.01 ± 1.04 cm) wasachieved on DKW medium containing 2.5 mg L-1

BAP.

The experiment with sycamore explants from invitro seedlings was not successful. With theexception of callus formation at the base of theexplants there was a general lack of response tothe culture conditions and to a range of media andhormone concentrations (results not presented).The inclusion of activated charcoal in the mediumdecreased the level of callus formation, asobserved in ash, but in the case of sycamore it didnot have any effect on shoot growth ormultiplication.

Difficulties in getting sycamore to proliferate andgrow in vitro have been a common observation ofmany researchers. Many approaches have beenattempted on a range of Acer species but successhas been rarely reported. Hanus and Rohr (1985)obtained in vitro plants from stem explants of Acerpseudoplatanus L. and A. negundo using MS basalsalts with Bourgin and Nitsch (1967) organiccomponents and activated charcoal. Attempts torepeat their work during this project did not meetwith success.

The success achieved with juvenile ash tissue isconsistent with the few reports in the literature.Generally, success with Fraxinus species has beenlow. Preece et al. (1987) obtained plantlets fromsterilised buds of F. americana L. (white ash)seedlings. Chalupa (1990) reported shootproliferation of ash explant from seedling on MSand DKW media supplemented with BAP andIBA. Regeneration of shoots from embryohypocotyls has been reported by Tabrett andHammatt (1992). Hammatt and Ridout (1992)recommended DKW medium supplemented with22.2 µM BA (benzyl adenine) as an appropriatemedium for the micropropagation of common ash.Such reports are not inconsistent with the resultsreported here.

37

BAP(mg L-1)1.02.55.0

Mean shootmultiplication

1.33b1

1.62a1.77a

Mean shoot height mm14b16a10c

Mean callus formation

0.78b1.56a1.45a

TABLE 5: EFFECT OF DIFFERENT CONCENTRATIONS OF BAP ON SHOOT MULTIPLICATION, SHOOTHEIGHT AND CALLUS FORMATION AT THE BASE OF ASH NODAL EXPLANTS.

1Means followed by the same letter are not significantly different, (p ≤ 0.05).

36

cultures. Conventional disinfection methods wereunsuccessful, since plant tissues were damaged bylong exposure time to sterilants, which wasnecessary to remove exogenous contaminants.Endogenous bacteria and fungi (e.g. Rhizoctonia,Alternaria and Stilbella) could not be eliminatedby surface sterilisation of plant material.

Hammatt (1996) reported that induction of old ashmaterial from mature ash trees into culture washampered by difficulties in surface sterilisation ofthe initial explants. In the trials reported here,various combinations of treatments failed toproduce uncontaminated cultures. In oneexperiment, only two ‘clean’ cultures wereobtained from 120 initial explants of one clone.Even then, contaminating Bacillus appeared fromthese after four months.

Part of the reason for the severe contaminationmay have been associated with the time of year inwhich the cuttings were taken (January-February).It is widely reported that shoots taken during thewinter months become severely contaminated anddisinfection is particularly difficult.

Selection of explant material in the properphysiological condition may help to ensuresuccessful culture initiation. It is commonlyaccepted that actively growing shoot tips are thebest explants to use for initiating shoot cultures(Kyte 1987). Shoot tips collected at various timesduring the spring growth flush, variedconsiderably in their ability to initiate proliferatingshoot cultures. Experiments used flushed budsfrom dormant material in January and Februaryand shoot tip cultures in April. Shoot tip culturestaken in April suffered from both bacterialcontamination and the production of phenoliccompounds, leading to explant death. By takingexplants in May to June and modifying thedisinfection procedure, the success rate wasimproved but without totally eliminating theproblems.

The results of these experiments confirmed thatpre-washing of plant materials with a fungicide isnecessary. Surface sterilisation with calciumhypochlorite resulted in more clean explants withless damage and in addition production of phenoliccompounds decreased in the culture medium.

3.2 Shoot growth and multiplication of ash andsycamore in vitro

Having established material from a variety ofsources during the initial phases of the project, thenext objective was to achieve multiplication of thematerial. The accepted best practice in the case ofwoody plant propagation is to avoid any callusproduction and to encourage the development ofaxillary buds. From the viewpoint of achievingmultiplication, an option is to achieve extensiongrowth of the shoot and to then subdivide the shootinto nodal cuttings. A better option from amultiplication point of view is to achieve multiplebud and shoot development within the axillarybud. Under mixotrophic conditions, this isachieved by including plant hormones in themedium at low concentrations. Experience withautotrophic micropropagation is that conditionsthat are successful in conventionalmicropropagation rarely translate directly toautotrophic micropropagation.

The experimental work in this section lookedinitially at conventional multiplication methodsusing juvenile material under mixotrophicconditions. This was then expanded to examinethe effect of high light and carbon dioxideenrichment and finally the use of cuttings frommature trees under autotrophic conditions.

Type of disinfectant

DomestosCalcium hypochloriteSodium hypochloriteMilton

Fungal contamination

20a1

0b0b0b

Phenolic compounds

53ab33c48b61a

Non-damaged explants

24b39a27b27b

1Means followed by the same letter are not significantly different, (p ≤ 0.05).

TABLE 4: EFFECT OF DISINFECTANT TYPE ON FUNGAL CONTAMINATION, OCCURRENCE OF NON-DAMAGED EXPLANTS AND PRODUCTION OF PHENOLIC COMPOUNDS ON SYCAMORE EXPLANTS.

%

medium with hortifoam. DKW with vermiculiteor vermiculite plus gelrite and WPM with agar,hortifoam and vermiculite plus gelrite did not havesignificantly fewer roots (Table 10). There weresignificant interactions between the conductivityand the type of medium and the percentage ofrooted explants with WPM at a conductivity of 3.2dS m-1, yielding 58% rooted explants and DKW atfull strength (6.1 dS m-1) yielding 32% rootedexplants (Table 11).

The interaction of medium by substrate byconductivity reveals that the type of substrate hadthe greatest effect on the number of rooted plantsand the effect of high conductivity was mitigatedby the type of substrate. In general the highestnumber of rooted plants was observed on the Enshimedium with foam (92% rooted), and DKW withvermiculite plus gelrite (88% rooted), while thelowest was on DKW with foam (Table 12).

3.2.2.3 Number of roots per plantThe type of medium and the interaction of mediumand substrate and medium and conductivity hadsignificant effects on the number of roots perplant. The maximum number of roots per plantwas observed on the WPM and DKW media whichwere significantly higher than those produced onthe Enshi medium (Table 7).

Explants placed on Enshi medium produced amaximum number of roots per explant when foamwas used as the substrate. The lowest root numberper plant was observed in DKW with foam (Table 10). The number of roots per plant was ata maximum in the WPM with a conductivity of 2.0 dS m-1 and minimum in full strength DKW(Table 11).

3.2.2.4 Length of rootsThe type of substrate and the interaction ofmedium and substrate, substrate and conductivityand medium by substrate by conductivity had

significant effects on the root lengths of ashexplants. The most significant root length wasobserved on media in which agar was used as thesubstrate. The shortest root lengths were producedin hortifoam (Table 8) and the combination ofDKW with hortifoam (Table 10).

Roots in the Enshi medium with agar or hortifoamwere significantly longer than those produced inthe vermiculite or vermiculite plus gelritetreatments. With WPM, when agar was used as asubstrate, the root length was significantly longerthan in hortifoam, vermiculite, or vermiculite plusgelrite. In the DKW with agar treatment, fullstrength medium reduced the root lengthcompared with the length at lower conductivities.DKW with vermiculite or vermiculite plus gelriteat full strength (EC = 6.1) produced the longestroots (Table 12).

3.2.2.5 Root fresh weightThe type of medium, substrate and conductivityhad significant effects on the root fresh weightproduced by the explants. DKW was the bestmedium for maximising root fresh weight. Thelowest root fresh weight was observed in the Enshimedium, which was significantly lower than WPMor DKW media (Table 7). Root fresh weight washighest when agar was used as a substrate and itwas significantly different from other substrates.Root fresh weight was lowest when foam was usedas a substrate (Table 8). Root fresh weight wassignificantly higher in full strength medium(highest level of EC for each medium) than thosewith a lower conductivity (Table 9).

3.2.2.6 Shoot growth (height)The type of medium, substrate and conductivityhad significant effects on shoot height of ashexplants. The maximum shoot height wasobserved in the WPM and DKW media and thesewere significantly higher than on Enshi medium(Table 7).

39

Substrate

AgarHortifoamVermiculiteVermiculite + gelrite

Survival %

98.0a1

80.5b78.6b92.0a

Shoot heightcm

2.11c2.44bc3.58a2.65b

Root lengthcm

5.72a2.47c3.37b3.74b

SHFWg

0.25b0.23b0.36a0.19b

RFWg

0.15a0.06c0.10b0.07c

1Means followed by the same letter are not significantly different, (p ≤ 0.05). SHFW: shoot fresh weight, RFW: root fresh weight.

TABLE 8: EFFECT OF SUBSTRATE ON SURVIVAL, SHOOT HEIGHTS, ROOT LENGTHS AND SHOOT ANDROOT FRESH WEIGHTS OF ASH EXPLANTS.

3.2.2 Effects of CO2

enrichment, basal media,conductivity levels and supporting matrix onphotoautotrophic growth of ash explants in vitro.

The objective of this experiment was to find thebest substrate and medium with an appropriateelectrical conductivity for photoautotrophicculture of ash nodal explants. The experimentconsisted of a factorial design using three differentmedia (WPM, DKW and Enshi) at four differentconductivities (full strength and diluted to give ECreadings of 2.5, 2.0 and 1.5 dS m-1) and fourdifferent substrates {agar, hortifoam, vermiculiteand vermiculite plus gelrite (1.5 g L-1)}. Fivereplicates per treatment were used each containingfive explants. Nodal explants from a single cloneof in vitro grown seedlings were placed on shootmultiplication medium (DKW with 5 ppm BAP,30 g L-1 sucrose and 7 g L-1 agar) in 60 ml glass jarsfor one week prior to transferring to theappropriate treatment in Magenta vessels withvented GA-7 lids. The cultures were placed in thephotoautotrophic growth chamber with a PPFD of90 µmol s-1m-2, 3000 ppm CO

2, a 16 hr-photoperiod

at 25 ˚C. The survival rate, number of rootedexplants, the number of roots, root lengths, rootfresh weights, shoot elongation and shoot freshweights were measured after six weeks.

3.2.2.1 Survival rateType of medium, substrate, interaction of mediumand substrate, medium and EC, medium andsubstrate and EC all had significant effects on thesurvival rate of plants. In general, the survival ratewas significantly higher on Enshi medium than oneither WPM or DKW media (Table 7). Survival

rate was highest on agar and vermiculite, the lattertwo substrates being significantly poorer (Table 8).

The lowest survival rate was on WPM with aconductivity of 2.5 dS m-1 (Table 11). Maximumsurvival rate was observed in Enshi medium withno significant differences between the differentconductivities and substrates. The minimumsurvival rate of explants was observed in WPMand DKW with agar or vermiculite plus gelrite. Ingeneral, the treatments with foam and vermiculitedried out faster than those with agar, orvermiculite plus gelrite and therefore the survivalrates decreased on these substrates. Thedeleterious effect of high conductivity in fullstrength WPM and DKW was most severe in thehortifoam substrate followed by vermiculite(Table 12). The naturally high conductivity wasprobably exacerbated by evaporation from themedium, leading to still higher concentrations ofions. In addition, it is well known that agar (andpresumably gelrite) bind a large proportion of theions in the medium in a manner that prevents themfrom having an effect on the plant tissue. Thiswould mediate the effect of high ionicconcentrations when such gelling agents are used.

3.2.2.2 Rooting of ash nodal explantsThe type of medium and the interactions betweenmedium and substrate and conductivity ofsubstrate had significant effects on the percentageof rooted explants. The percentage of rootedexplants was maximised in WPM and there weresignificant differences in this regard betweenWPM and Enshi, or DKW (Table 7). Themaximum rooting of plants occurred on Enshi

38

Medium

With ACWithout AC

Mean shootmultiplication

1.00b1

2.19a

Mean shoot height (mm)0.9b1.7a

Mean callus formation

0.09b2.43a

TABLE 6: EFFECT OF ACTIVATED CHARCOAL ON SHOOT MULTIPLICATION, SHOOT HEIGHT ANDCALLUS FORMATION AT THE BASE OF ASH NODAL EXPLANTS.

1Means followed by the same letter are not significantly different, (p ≤ 0.05).

Medium

EnshiWPMDKW

Survival%

98a1

82b90b

Rooting %

42.3b56.4a46.2b

No of rootsper plant

1.37b1.82a1.65a

Shoot heightcm

2.24b2.97a2.88

SHFWg

0.21b0.23b0.34a

RFWg

0.074b0.10a0.11a

TABLE 7: EFFECT OF MEDIUM ON SURVIVAL, ROOTING, NUMBER OF ROOTS PER PLANT, SHOOTHEIGHT AND SHOOT AND ROOT FRESH WEIGHT OF ASH EXPLANTS.

1Means followed by the same letter are not significantly different, (p ≤ 0.05), SHFW: shoot fresh weight, RFW: root freshweight.

experiment indicate successful rooting of ashexplants without sucrose or hormones with ahigher percentage rooting than has previouslybeen reported in conventional in vitro culture. Inaddition, losses due to contamination wereminimal.

3.2.3 Effects of kinetin and NAA on rooting, budbreak and shoot growth of ash cuttings

The work reported to date on shoot growth andproliferation has used nodal cuttings of juvenilematerial derived from in vitro germinated seeds.The reason that the majority of the publishedliterature has used material from this source isbecause it is possible to obtain contamination-freecultures and it appears more amenable tomanipulation of its growth pattern by the use ofplant growth regulators. According to Hammatt(1996) it took 200 to 600 days after initiation ofmature ash material for 42% of the shoots to rootand this was accompanied by 100% contaminationwith Bacillus. In order to obtain shoot materialsuitable for in vitro culture or direct establishmentof ash for microcoppicing in the PAM system,several experiments studying the effect ofdifferent media, substrate and plant growthregulators were carried out.

This experiment was part of a series examining theeffects of a range of hormone medium andsubstrate combinations in an attempt to rootmature cuttings of ash taken in September. In thisparticular study the effect of kinetin and NAA andSolufeed medium on rooting, bud break and shootgrowth of ash cuttings was examined. TheSolufeed medium supported bud break andhealthy shoot growth (Table 13). No rooting ofthe cuttings occurred, even though callusing of thebasipetal end occurred in many of the explants.

3.2.4 Effect of BAP and NAA on bud break andshoot growth of ash cuttings

The objective of this experiment was to study theeffect of full and half strength DKW supplementedwith BAP and NAA and combinations of BAP andNAA on rooting, bud break and shoot growth.Ash cuttings with one or two buds were takenfrom old trees in late August 1997 and cultured inbaby jars containing full or half strength DKWwithout sugar and solidified with agar.

The highest number of bud breaks and greatestshoot growth was observed on media with 2.0 mg

L-1 BAP. Although this treatment was significantlydifferent to the control, there was no significantdifference between 2.0 mg L-1 BAP and otherplant growth regulators used in this experiment(Table 14). No rooting of the cuttings occurredduring the period of the experiment.

3.2.5 Effect of auxin on rooting and shoot growthof ash semi-hardwood cuttings

The objective of this experiment was to investigatethe effect of IBA and NAA on rooting of ash semi-hardwood cuttings. Cuttings were taken on the 8thof July 1997 from mature trees. Solufeed at 3.0 gL-1 was supplemented with different concentrationsof NAA (0.00, 0.25, 0.50 and 1.00 mg L-1 ) and IBA(0.00, 0.25, 0.50 and 1.00 mg L-1). Hortifoam wasused as the substrate.

Treatment of cuttings with differentconcentrations of IBA and NAA was not effectiveon rooting of cuttings, but bud break and shootgrowth was significantly improved. NAA wassignificantly more effective than IBA on budbreak (Table 15).

3.2.6 Effect of hormone and type of cuttings onbud break and shoot growth of ash

Experiments were carried out with semi-hardwoodcuttings taken in the months of August andSeptember. Cuttings were subjected to a widerange of media, hormones (both singly and incombination) and substrates. Results suggestedthat while this material can be introduced toculture under photoautotrophic conditions andwhile bud break and some shoot elongation can beachieved, the lack of rooting ability ultimatelycompromises the sustainability of such material inculture.

A further experiment was set up comparing theresponse of semi-hardwood cuttings from juveniletrees and from mature trees with softwood cuttingstaken from epicormic shoots removed frommature trees on Belfield campus. The type ofcutting and the interaction between hormone andtype of cutting had significant effects on budbreak. Bud break and shoot growth of cuttingsfrom epicormic shoots and young trees weresignificantly higher than on cuttings taken fromold trees. There was no significant differencebetween cuttings taken from epicormic shoots andyoung trees.

41

The greatest shoot height was observed in thetreatments in which vermiculite was used as asubstrate and the lowest shoot height was observedin the media in which agar was the substrate(Table 8). There was a correlation betweenmedium strength and shoot height with maximumshoot height occurring in the full strength mediaand the minimum shoot height in the media with aconductivity of 1.5 dS m-1 (Table 9).

The interaction of medium and substrate had asignificant effect on the shoot height of ashexplants, as with most of the other variablesexamined The WPM and DKW media withvermiculite resulted in the greatest shoot heights inthe trial. The minimum shoot height was observedon Enshi medium with agar (Table 10).

3.2.2.7 Shoot fresh weightMedium, type of substrate, the conductivity of themedium and the interaction between medium andconductivity had significant effects on shoot freshweight. DKW medium yielded the maximumshoot fresh weight and this was significantlyhigher than on Enshi or WPM media (Table 7).Shoot fresh weight was highest when vermiculitewas used as a substrate and it was significantlybetter than the three other substrates used(Table 8).

Shoot fresh weight was highest in full strengthmedia (the highest EC) and it was significantlylower with the lower levels of conductivity. Shootfresh weight was lowest at the lowest level ofconductivity (EC = 1.5 dS m-1) but it was notsignificantly different to that achieved at theintermediate levels (EC = 2.5 or 2 dS m-1) (Table 9).

3.2.2.8 Discussion of the effect of CO enrichment,basal media, conductivity level and supportingmatrix on photoautotrophic growth of ash explantsin vitro

Ash explants exhibited strong and vigorousgrowth on a range of media under CDE conditionswithout sugar and hormones in the media. Due tothe wide range of interactions between the variousfactors, generalisations and drawing firmconclusions can be difficult. However, in general,Enshi medium was good for survival and rootingof ash explants, even though root growth was notas vigorous as with other salt mixtures. Shootgrowth on Enshi, while adequate was not asvigorous as with DKW medium. High levels ofconductivity tended to decrease rooting and rootgrowth but increased shoot growth. The effect ofsubstrate was highly interactive with conductivityand type of medium. Hortifoam was excellent forgrowth with Enshi medium but not with DKW.Agar maximised root length but not rootingfrequency or number of roots and there was alsothe problem of functionality of roots produced inagar (absence of root hairs and lack of oxygen inthe substrate) and the fact that long roots tend toget damaged in the transplanting process.Vermiculite is a good compromise substrate but isnot very user friendly under laboratory conditionsand has a tendency to dry out, as does hortifoam,in longer-term cultures.

In agreement with our results, Chalupa (1987)reported that low salt media such as WPMstimulated root development in woody plantsmore than the high salt MS medium. Chalupa(1990) reported between 62 to 84% rooting ofshoots on WPM containing sucrose and auxin.Differences were observed in the rooting ofdifferent clones. Hammatt (1996) reported callusformation and very poor root growth in ashexplants. To overcome this they exposed theirexplants to 21 days auxin treatment followed bytransfer to an auxin-free medium. Results of this

40

ECdS m-1

Full strength2.52.01.5

Shoot heightcm

3.08a1

2.69ab2.54b2.47b

SHFW g

0.34a0.28ab0.23b0.19b

RFW g

0.13a0.10ab0.07b0.08b

TABLE 9: EFFECT OF CONDUCTIVITY ON SHOOT FRESH WEIGHT AND ROOT FRESH WEIGHT OF ASHEXPLANTS.

1Means followed by the same letter are not significantly different, (p ≤ 0.05).SHFW: shoot fresh weight, RFW: root fresh weight.

2

43

Medium

EnshiEnshiEnshiEnshiEnshiEnshiEnshiEnshiEnshiEnshiEnshiEnshiEnshiEnshiEnshiEnshiWPMWPMWPMWPMWPMWPMWPMWPMWPMWPMWPMWPMWPMWPMWPMWPMDKWDKWDKWDKWDKWDKWDKWDKWDKWDKWDKWDKWDKWDKWDKWDKW

Substrate

AgarAgarAgarAgarHortifoamHortifoamHortifoamHortifoamVermiculiteVermiculiteVermiculiteVermiculiteVermiculite + GVermiculite + GVermiculite + GVermiculite + GAgarAgarAgarAgarHortifoamHortifoamHortifoamHortifoamVermiculiteVermiculiteVermiculiteVermiculiteVermiculite + GVermiculite + GVermiculite + GVermiculite + GAgarAgarAgarAgarHortifoamHortifoamHortifoamHortifoamVermiculiteVermiculiteVermiculiteVermiculiteVermiculite + GVermiculite + GVermiculite + GVermiculite + G

ECdS m-1

2.412.52.01.52.412.52.01.52.412.52.01.52.412.412.01.53.22.52.01.53.22.52.01.53.22.52.01.53.22.52.01.56.112.52.01.56.112.52.01.56.112.52.01.56.112.52.01.5

Survival%

100a1

100a100a96ab

100a100a100a100a100a96ab

100a92ab

100a96a88ab

100a100a100a88ab

100a76b68bc92ab72bc80ab52c56bc84ab96ab52c96ab

100a96ab

100a100a100a44c98ab32c92ab76b64bc84ab68bc84ab

100a100a92ab

Root lengthmm

7.3a6.0ab3.3bc5.6b5.0b4.1b3.8bc4.4b3.3bc0.6d2.0c2.6c1.0c3.9b1.9cd2.6c6.3ab7.4a3.7bc7.2a1.7cd1.8c2.7c1.9c4.6b2.0c3.2bc2.9c4.2b3.8b4.3b2.9c3.5bc6.5a5.3b6.5a

0d2.9c0.5d0.8d7.3a4.8b4.7b2.4bc7.6a4.7b3.8b4.2b

Rooting%52c8e8e

24d52c72b92a68b56c12d40cd44c16e44c28d22d80a60b36d60b64b60b62b40cd44c32d48c80a80a28d68b60b36d32d28d32d0e

34d12e36cd60bc44c72b52bc44c68b72b88a

1Means followed by the same letter are not significantly different, (p ≤ 0.05), G: gelrite.

TABLE 12: EFFECT OF MEDIUM, SUBSTRATE, AND EC ON SURVIVAL, ROOTING AND ROOT LENGTHOF ASH EXPLANTS.

42

Medium

EnshiEnshiEnshiEnshiWPMWPMWPMWPMDKWDKWDKWDKW

Substrate

agarhortifoamvermiculitever + gelriteagarhortifoamvermiculitever + gelriteagarhortifoamvermiculitever + gelrite

Survival %99a1

100a96a96a96a76b68b86b99a66b72b

94ab

Rooting%

33c71a38bc30c58ab57ab51b59ab32c28c57ab68a

No. of rootsper plant

1.21b1.82a1.26b1.17b1.72a1.76a1.75a2.05a1.561.01b1.97a2.06a

Shoot heightcm

1.69d3.31b2.12cd1.85cd2.54c2.18cd4.18a2.96bc2.11cd1.83d4.44a3.14b

Root lengthcm

5.55ab4.32b2.12c2.37c6.15a2.03c3.19bc3.80bc5.45ab1.05d4.79b5.04ab

1Means followed by the same letter are not significantly different, (p ≤ 0.05), ver : vermiculite.

1Means followed by the same letter are not significantly different, (p ≤ 0.05).SHFW= shoot fresh weight.

TABLE 10: EFFECT OF MEDIUM AND SUBSTRATE ON SURVIVAL ROOTING, NUMBER OF ROOTS/PLANT,SHOOT HEIGHT AND ROOT LENGTH OF ASH EXPLANTS.

TABLE 11: EFFECT OF MEDIUM AND CONDUCTIVITY ON SURVIVAL, ROOTING, NUMBER OFROOTS/PLANT AND SHOOT FRESH WEIGHT OF ASH EXPLANTS.

Medium

EnshiEnshiEnshiEnshiWPMWPMWPMWPMDKWDKWDKWDKW

EC dS m-1

2.42.52.01.53.22.52.01.56.12.52.01.5

Survival %

100a1

98ab97ab97ab88bc68bc82.5bc88.5ab74bc90ab78.5bc88bc

Rooting%

33b71a38ab30b58a57a51a59a32b28b57a68b

No. of roots per plant

1.33b1.36b1.28b1.49a1.92a1.71a2.05a1.61a1.22b1.72a1.75a1.91a

SHFWg

0.21c0.21c0.21c0.20c0.32bc0.24c0.21c0.19c0.52a0.23c0.43ab0.17c

subject to conditions that caused bud break andelongation. These were excised, treated with anIBA or NAA rooting powder and placed in eithervermiculite, hortifoam or agrifoam slabs inenclosed containers (i.e. not a recirculatingnutrient system).

Sycamore cuttings rooted readily in the agrifoamslabs and extension growth of the cuttingsoccurred. After approximately six weeks, apicalcuttings were removed, leaving at least two nodalbuds on the original cutting. Bud break andgrowth occurred within a further four weeks, whencuttings were again removed. This procedure wasrepeated over a five month period, with thecuttings being re-rooted into further agrifoamslabs. The growth of sycamore in the stool-bedsestablished in the re-circulating system wassuperior to that in the enclosed system. There areprobably a number of factors influencing this,including the ability to maintain the conductivityof the medium in the case of the recirculatingsystem and the improved ventilation in this systemleading to a more ‘normal’ type of shoot growth.The softwood microcuttings could be easily rootedunder photoautotrophic conditions in vitro and thecoppice beds yielded up to four microcuttings perplant after the third cycle of cutting. It wasimportant not to stress the plants in themicrostoolbed as this led to leaf loss and slowingof shoot growth, or in the worst case, induction ofdormancy.

Initial rooting of the ash microcuttings was quitepoor with only 30% rooting achieved. Withsuccessive pruning (micro-coppicing) of the

cultures however, this increased to 90% success ina five week period as opposed to the 12 weekstaken to root the initial cuttings. After eachpruning, bud break and stem elongation took 17 to21 days, after which each stem had produced afurther two to three nodes and the material wasready for a further pruning. The cuttings took 10to 14 days for visible root initials to appear andhad a well-developed root system by five weeksafter subculture. The initial multiplication ratewas one microcutting per plant but by repeatedpruning the growth of more lateral shootsoccurred, to yield two to three cuttings per plantby the fourth and fifth pruning. The trial wasterminated after five successive sets of cuttingshad been taken.

3.2.9 General discussion of shoot growth andmultiplication in ash and sycamore

Direct establishment of sycamore nodal or shoottip cuttings using plant growth regulators either byinclusion in the medium or as a short exposure(quick dip) treatment displayed differing degreesof success depending on the age of the stock plantand the season in which the cuttings werecollected. More than 70% of cuttings collectedfrom one year old glasshouse grown materialrooted under CDE conditions to establish newplants, whereas this figure dropped to 40% incuttings from newly flushed buds from fieldgrown plants. Substrate had significant effects,with both hortifoam and vermiculite provinguseful with results of 83% and 77% rootingrespectively.

45

Medium

DKWDKWDKWDKWDKWDKW1/2DKW1/2DKW1/2DKW1/2DKW1/2DKW1/2DKW

Plant growth regulator

ControlNAA 0.2 mg L-1

NAA 0.5 mg L-1

BAP 2.0 mg L-1

BAP 2.0 mg L-1+NAA 0.02 mg L-1

BAP 2.0 mg L-1+NAA 0.2 mg L-1

ControlNAA 0.2 mg L-1

NAA 0.5 mg L-1

BAP 2.0 mg L-1

BAP 2.0 mg L-1+NAA 0.02 mg L-1

BAP 2.0 mg L-1+NAA 0.2 mg L-1

Bud break% 13

713333

1313

7201

202

272

1313

Shoot growthmm3.501.505.00

12.002

5.003.001.00

11.502

11.501

5.502.001.50

TABLE 14: EFFECT OF PLANT GROWTH REGULATORS AND FULL OR HALF STRENGTH DKW ON BUDBREAK AND SHOOT GROWTH OF ASH CUTTINGS.

1 p ≤ 0.05 2 p ≤ 0.001 3 p ≤ 0.0001.

Bud break in cuttings taken from mature trees onthis occasion was negligible (Table 16). Rootingwas not observed in any of the treatments withinthe time scale of the experiment.The interaction between source of cutting andhormone treatment is shown in Table 17.

The level of bud break was zero or extremely lowin nodal explants from mature trees irrespective ofthe hormone treatment applied. With explantsfrom a juvenile source, 40% of the controlexplants exhibited bud break. Hormone treatmentdid not significantly improve this rate, indeedBAP at 3.0 ppm significantly decreased the rate ofbud break. While the no hormone control withepicormic shoots was not significantly different tothe juvenile source of explants, it was significantlybetter than direct cuttings from mature trees inrespect of bud break and subsequent shoot growth.Shoot growth in the epicormic control was lowerthan that from the juvenile control, reflectingperhaps the greater carbohydrate reserves in thelarger cuttings from the juvenile trees.

3.2.7 Effect of TDZ, BAP and GA3on bud break

and shoot growth of dormant sycamore buds

The objective of this experiment was to obtainshoot explants for conventional tissue cultures andstudy the possibility of direct establishment ofdormant material cultured on agrifoam under CDEconditions. Hardwood cuttings of sycamore withone or two dormant buds were taken from old treeson the 17th of December, 1997 and surface

sterilised. The cuttings were treated with TDZ (0,25, 50 and 100 ppm), BAP and GA

3(0, 125, 250,

5000 and 1000 ppm) for 0, 1, 3, 6 and 24 hours.Cuttings were cultured in baby food jars ventilatedwith Sun Caps, containing DKW medium,Solufeed with 6.0 g L-1 agar, without sugar andplaced under CDE and high PPFD conditions.The effect of plant growth regulators,concentrations, duration of hormone treatmentsand the interaction between hormones andconcentrations on bud break were significant. Ingeneral, treatment with GA

3induced significantly

more bud break than TDZ or BAP (Table 18).

Long (24 hour) exposure times to PGRs invariablyled to no bud break, except in the case ofgibberellic acid treatment where significantlyimproved rates of bud break were observed (Table 19).

3.2.8 Establishment of in vitro stool beds

Stool beds were established for both sycamore andash. In the case of sycamore, the route was directestablishment of semi-hardwood cuttings inAgrifoam slabs (90 x 1300 x 25 mm). These wereplaced either in an enclosed system in containerswith ventilation and placed on the growth roomshelf under photoautotrophic conditions, or theywere placed in a hydroponic system withrecirculating nutrients, again in the growth roomunder photoautotrophic conditions.In the case of ash, microcuttings were removedfrom semi-hardwood cuttings that had been

44

Hormone

ControlNAA (0.2 mg L-1)NAA (0.5 mg L -1)Kin (0.5 mg L-1)Kin (0.5 mg L-1) + NAA (0.01 mg l-1)Kin (0.5 mg L-1) + NAA (0.1 mg l-1 )Kin (1.0 mg L-1)Kin (1.0 mg L-1) + NAA (0.01 mg L-1)Kin (1.0 mg L-1) + NAA (0.1 mg L-1)Kin (2.0 mg L-1)Kin (2.0 mg L-1) + NAA (0.01 mg L-1)Kin (3.0 mg L-1)Kin (3.0 mg L-1) + NAA (0.01 mg L-1)Kin (3.0 mg L-1) + NAA (0.1 mg L-1)

Bud break %442

22402

331

533

402

603

502

402

472

673

533

583

733

Shoot growth(mm)

6.62

5.06.62

8.73

10.33

5.51

8.03

5.01

3.05.51

5.51

7.83

3.712.03

TABLE 13: EFFECTS OF KINETIN AND NAA ON BUD BREAK AND SHOOT GROWTH OF ASH HARDWOODCUTTINGS.

1 p ≤ 0.05 2p ≤ 0.001 3p ≤ 0.0001, Kin: kinetin.

47

TABLE 19: EFFECT OF EXPOSURE TIME, PGR TYPE AND CONCENTRATION ON BUD BREAK OFDORMANT SYCAMORE BUDS.

PGR

TDZTDZTDZTDZTDZTDZTDZTDZTDZTDZTDZTDZTDZTDZTDZTDZBAPBAPBAPBAPBAPBAPBAPBAPBAPBAPBAPBAPBAPBAPBAPBAPGA3GA3GA3GA3GA3GA3GA3GA3GA3GA3GA3GA3GA3GA3GA3GA3

Conc. (mg L-1)

0000

2525252550505050

100100100100

0000

250250250250500500500500

1000100010001000

0000

250250250250500500500500

1000100010001000

Time(hr)136

24136

24136

24136

24136

24136

24136

24136

24136

24136

24136

24136

24

Bud break%60474007

27200

1300000

470

6747400

1720707

2000

2737130

6047270

3340533373808033

1001004733

p ≤

0.00010.0010.05n.s.n.s.0.05n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.0.001n.s.0.00010.0010.05n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.0.050.05n.s.n.s.0.00010.0010.05n.s.0.050.050.00010.050.00010.00010.00010.050.00010.00010.0010.05

n.s: not significant.

46

Auxin

NAAIBA

Bud break %

52.501

31.25

Shoot growthmm

10.811

6.50

TABLE 15: EFFECT OF IBA AND NAA ON BUD BREAK AND SHOOT GROWTH OF ASH SEMI-HARDWOODCUTTINGS.

TABLE 16: EFFECT OF AGE OF STOCK PLANT ON BUD BREAK AND SHOOT GROWTH OF ASH.

TABLE 17: EFFECT OF SOURCE OF CUTTING AND HORMONE ON BUD BREAK OF ASH CUTTINGS.

1 p ≤ 0.0001

Type of cutting

MatureJuvenileEpicormic

Bud break %4301

401

Shoot growth mm0.34.51

5.01

Source of cutting

MatureMatureMatureMatureMatureJuvenile JuvenileJuvenileJuvenileJuvenileEpicormicEpicormicEpicormicEpicormicEpicormic

Hormone

ControlNAA (0.5 mg L-1)kin (0.5 mg L-1) kin (2.0 mg L-1)BAP (3.0 mg L-1)ControlNAA (0.5 mg L-1)kin (0.5 mg L-1) kin (2.0 mg L-1)BAP (3.0 mg L-1)ControlNAA (0.5 mg L-1)Kin (0.5 mg L-1) Kin (2.0 mg L-1)BAP (3.0 mg L-1)

Bud break%0

1000

10405020400

3020603060

Shoot growthmm0.01.00.00.00.57.0

10.11.53.50.03.05.53.52.58.0

Hormone

TDZBAPGA

3

Bud Break%

16.2418.7452.491

1 p ≤ 0.0001

TABLE 18: EFFECT OF HORMONE TREATMENT ON BUD BREAK OF DORMANT SYCAMORE BUDS.

1 p ≤ 0.0001

harvests from the stool beds however, the rootingpercentage increased up to 90% and new cuttingscould be harvested at 3 to 4 week intervals.

3.3 Rooting and weaning

The final stage of any micropropagationprogramme is the rooting and weaning of theshoots back into in vivo conditions. Much of thework on establishment of cultures underphotoautotrophic conditions carried out during thecourse of the project involved the rooting ofvarious explants on different substrates, so asubstantial body of knowledge was built up in thisregard and has been reported in previous sections.In general terms both soft and semi-hardwoodcuttings from sycamore could be rooted with ease.Ash semi-hardwood cuttings could not be rootedbut bud break and stem elongation could beachieved, providing a source of softwood cuttings.Trials were carried out examining the effects ofdifferent hormone treatments on the rooting ofsoftwood cuttings, which are the ultimate productof any micropropagation system.

3.3.1 Effect of IBA and NAA on rooting of ashsoftwood cuttings

Softwood cuttings which were regenerated fromstem cuttings collected from old trees under CDEconditions in hortifoam were treated with 0.5 mg L-1 IBA or NAA and cultured in Magenta boxeswith ventilated lids. Enshi medium was used withvermiculite as the substrate. There was nosignificant difference between IBA and NAA onpercentage rooting, which in this case was low.Untreated cuttings failed to root (Table 20).

3.3.2 Weaning of micropropagated material ofash and sycamore

There were no large-scale replicated trials onweaning of micropropagated plants carried outduring the course of this study. As plants becameavailable from various trials and experiments,small samples were potted on and placed in theglasshouse. All material of both ash and sycamoreweaned and grew with 100% survival rate undernormal glasshouse conditions. These findings arein full agreement with all other studies on weaningof micropropagated material from photo-autotrophic sources. Due to a fully operativephotosynthetic pathway, well-developed cuticle,functional stomata and functional roots, plantsderived from cultures under photoautotrophicconditions transferred to in vivo conditions muchmore readily than those from mixotrophicconditions did.

49

Treatment

ControlIBANAA

Rooting%0b1

28ab21ab

TABLE 20: EFFECT OF IBA AND NAA ON ROOTING OF ASH SOFTWOOD CUTTINGS.

1Means followed by the same letter are not significantly different, (p ≤ 0.05).

48

All cuttings rooted without the use of plant growthregulators, although plant growth regulatorsproved useful for bud break and enhancingsubsequent shoot growth. These results indicatedthat rooting softwood cuttings from sycamore maybe achieved without plant growth regulators underphotoautotrophic conditions and withoutcontamination losses and can be used as the basisfor setting up in vitro stool beds for themultiplication of selected clones.

Direct establishment of stem cuttings collectedfrom mature sycamore trees was successful with87% of semi-hardwood cuttings rooting whentreated with rooting hormone powder and culturedon hortifoam substrate. In general, hortifoamproved better than vermiculite as a substrate whenusing the quick dip method but vermiculite wassuperior when including PGRs in the growthmedium. Cold treatment of sycamore semi-hardwood cuttings did not increase the percentageof rooting unlike the reports for Acer palmatumatropurpureum cuttings collected in November(Marcinkowski 1988).

Semi-hardwood cuttings proved to be the mostsuitable type of sycamore cutting for the directestablishment and growth of non-axenic culturesunder photoautotrophic conditions. Stock plantsthat were heavily pruned in March produced newshoots, which were used for semi-hardwoodcuttings in July. The percentage of rootingincreased in these cuttings, confirming the resultsof Land et al. (1995) with American sycamore(Platanus occidentalis) where hedging stockplants was shown to rejuvenate cuttings.

Shoot elongation and further shoot initiation fromestablished cuttings was variable in the trialscarried out and application of different media didnot show a consistent response or pattern. Evenwithin treatments some buds developed vigorousextensions and growth rates while others remaineddormant. This phenomenon has been previouslyobserved in photoautotrophic woody plant culture(Long 2000). Sub-culturing only the most activelygrowing material leads over a number of cycles toa more homogeneous response pattern. Sycamoreis particularly sensitive to stress during culture andthis may be partly the cause of induced dormancyin the buds. It was noted that the leaves in newshoots produced under photoautotrophicconditions were red in colour and were slow togreen, suggesting perhaps light, temperature, oreven salt stress. All of these factors would be

worth investigating further. The commonly usedinorganic nutrients used in plant tissue culturehave been optimised for mixotrophic cultures, butnot photoautotrophic cultures. This point has beenstudied for photoautotrophic cell cultures (Hornand Widholm 1984) but not for micropropagationpurposes. Kozai et al. (1991) have pointed outthat the requirements of the two types of cultureshould be quite different. There is also someevidence to suggest that sycamore requires a lowlight intensity for shoot growth and perhaps thelevel of PPFD the cultures were subject to underthe stated conditions had a negative effect on shootgrowth.

In this study, cuttings collected from late June toOctober from mature trees all sprouted to differentdegrees, depending on the medium and substrateused. It is the first report that bud break and shootgrowth can be achieved during active growth ofash trees growing under natural conditions.Silveira and Cottignes (1993) reported that stemcuttings of ash from 4 to 7 year old trees growingunder natural conditions only sprouted when takenfrom dormant material. Apical buds taken duringperiods of shoot apical dormancy in September,January and March were able to sprout on avariety of media containing sucrose undermixotrophic conditions (Lloyd and McCown1980), but no growth was obtained from apicalbuds removed in May or June, which is the periodof cell proliferation and intense branch growth(Silveira and Cottignes 1993). In theseexperiments, bud break and sprouting could occurunder CDE and high PPFD conditions during allthe months in which cuttings were taken (June,July, August, September, November, December,January and March).

The type of medium had a significant effect onbud break, although at certain times of the year(March) bud break could be achieved by placingthe cuttings in water. Generally, Solufeed provedto be particularly efficacious. In terms ofsubstrate, in some trials hortifoam was clearlysuperior but in others agar or vermiculite were justas effective.

Microcuttings obtained from bud growth from arange of hardwood and semi-hardwood cuttingscould be rooted and used to establish in vitro stoolbeds. At this point only 30% rooting of suchcuttings had been achieved. This is consideredvery good as ash is considered very difficult topropagate from stem cuttings. In successive

51

5. REFERENCES

Boner, F. T. 1974. Fraxinus ash. Seeds of woodyplants in the United States. U.S.D.A. Agric.Handb. 450: 411-416.

Bourgin, J. P. and Nitsch, J. P. 1967. Productionof haploid Nicotiana from excised stamens. Ann.Physiology Vegetale 9: 377-382.

Chalupa, V. 1983. Micropropagation of coniferand broad-leaved forest trees. Commun. Inst. For.Czechoslovakia 13: 7-39.

Chalupa, V. 1987. European hardwoods. In Celland Tissue Culture in Forestry Vol. 3. Eds. Bonga,J. M. and Durzan, D. J. Martinus Nijhoff Publ.Dordrecht/Boston/Lancaster, pp 224-246.

Chapula, V. 1990. Micropropagation ofhornbeam (Carpinus betulus L.) and ash (Fraxinusexcelsior L.). Biol. Plant. 32:332-338.

Chalupa, V. 1993. Vegetative propagation of oak(Quercus robur and Q. petraea) by cutting andtissue culture. Annales des Sciente Forestieres 1:2958-3078.

Driver, J. A. and Kuniyuki, A. H. 1984. In vitropropagation of Paradox walnut rootstock. Hort.Science 19 (4): 507-509.

Hammatt, N. and Ridout, M. S. 1992.Micropropagation of common ash (Fraxinusexcelsior L.). Plant Cell, Tissue and OrganCulture 13: 67-74.

Hammatt, N. 1994. Shoot initiation in the leafletaxils of compound leaves from micropropagatedshoots of juvenile and mature common ash(Fraxinus excelsior L.). Journal of ExperimentalBotany 45 (275): 871-875.

Hammatt, N. 1996. Fraxinus excelsior L.(Common Ash). In Biotechnology in Agricultureand Forestry 35, Trees IV. Ed. Bajaj, Y. P. S.,Springer-Verlag, Berlin/Heidelberg/New York.

Hanus, D. and Rohr, R. 1985. Micropropagationde l’érable par bouturage in vitro de fragments degermination de trois espèces. Can. J. Bot. 63: 277-280. In Biotechnology in Agriculture andForestry, Vol. 5 Trees II. Ed. Bajaj, Y. P. S.,Springer-Verling, Berlin/Heidelberg/New York.

Hori, H. 1966. Gravel culture of vegetable andornamental crops. Agriculture and Horticulture:120.

Horn, M. E. and Widholm, J. M. 1984. Aspectsof photosynthetic cell cultures. In Applications ofGenetic Engineering to Crop Improvement. Eds.Collin, G. B. and Petolio, J. G., Martinus Nijhoff,Dordrecht, pp 113-161.

Kozai, T., Kubota, C. and Watanabe, I. 1988.Effects of basal medium composition on thegrowth of carnation plantlets in auto and mixo-trophic tissue culture. Acta Hort. 230: 159-166.

Kozai, T., Iwabuchi, K., Watanabe, K. andWatanabe, I. 1991. Photoautotrophic andphotomixotrophic growth of strawberry plantletsin vitro and changes in nutrient composition of themedium. Plant Cell, Tissue and Organ Culture25: 107-115.

Kyte, L. 1987. Plants from test tubes. TimberPress, Oregon.

Land, S. L. B. Jr., Elam, W. W. and Khan, M.1995. Rejuvenated sycamore cuttings for energyplantations. Biomass and Bioenergy 4: 255-264(In CAB Abstracts 1996-4/97).

Lloyd, G. and McCown, B. 1980. Commerciallyfeasible micropropagation of mountain laurel,Kalmia latifolia, by use of shoot tip culture.Combined Proceedings of the International PlantPropagators Society 30: 421-427.

Long, R. 1997. Photoautotrophicmicropropagation: A strategy for contaminationcontrol. In Pathogen and MicrobialContamination Management in Micro-propagation. Ed. Cassells, A. C., Developmentsin Plant Pathology 12: 267-278.

Long, R. 2000. Unpublished results. Green CropLtd., Carlow, Ireland.

Marcinkowski, J. 1988. Temperature pre-treatment of hardwood cuttings of ornamentaldeciduous shrubs. Acta Horticulturae 266 (1):363-367.

Murashige, T. and Skoog, F. 1962. A revisedmedium for rapid growth and bioassays withtobacco tissue culture. Physiol. Plant 15: 473 -497.

4. CONCLUSIONS

The overall aim of this project was to investigatethe potential of a photoautotrophic system forrapid clonal multiplication of ash and sycamore.While conventional mixotrophic micro-propagation of sycamore has been reported in theliterature, micropropagation of ash, except fromseedling tissue, has not been reported.

In the course of this project, it was demonstratedthat material from mature trees can be selected inthe field and introduced into culture in a mannerthat proved possible to use nodal cuttings toestablish micro-coppicing beds directly, usingsimple nutrient media. Shoots were rooted into arange of substrates other than agar, theconventional substrate for micropropagation.From a practical point of view, foam substratesproved easy to use, were autoclavable, could beused a number of times and yielded good results.With ash, direct rooting of cuttings was notpossible under the stated conditions and indeedthis confirms reports in the literature. However, asystem of inducing bud break and elongation ofnodal buds was developed and it proved possibleto root these shoots with a 30% success rateinitially. Once the material had been growingunder photoautotrophic conditions for a longerperiod, the success rate improved to 90%.

Proliferating nodal cultures of either ash orsycamore were not achieved. However, amultiplication system under autotrophicconditions was developed using nodal increases.This pathway is preferred in many herbaceouscrops due to the fact that it functions through thegrowth of single nodal buds, reducing theprobability of somaclonal variants, or mutantsoccurring. Under mixotrophic conditions, growthand nodal increase in woody plants can be slow,leading to the perception that proliferating nodalcultures, where axillary meristems within thenodal bud are encouraged to grow and developthrough the use of plant growth regulators, is amore efficient pathway for producingmicrocuttings. Under the autotrophic conditionsused in this study, sequential nodal cuttings couldbe removed from micro-coppices at three weekintervals, at a rate faster than can be achievedusing conventional cultures. In addition, ifcontamination and weaning losses are taken intoaccount it is probable that the multiplication ratesachieved under autotrophic conditions are equal tothose possible under mixotrophic conditions.

It is obvious that the techniques developed andexplored during the course of this project haveimmediate applications in the industrialpropagation of ash and sycamore.

50

53

Macro ElementsCaCl

2

Ca(NO3)

KH2PO

4

K2SO

4

MgSO4

7H2O

NH4NO

3

KNO3

NH4H

2PO

4

Micro ElementsCuSO

45H

2O

FeNa2

EDTAH

3BO

3

MnSO4

H2O

Na2MoO

4.H

2O

ZnSO4.7H

2O

CoCl2

6H2O

KIVitaminsGlycinemyo-InositolNicotinic AcidPyridoxine HClThiamine HCl

WPM

72.50386.80170.00990.00180.54400.00

0.2536.706.20

22.300.258.60

2.00100.00

0.500.501.00

DKW

112.501367.00265.00

1559.00361.49

1416.00

0.2544.634.80

33.800.39

17.00

2.00100.00

1.00

2.00

MS

332.02

170.00

180.541650.001900.00

0.02536.706.20

16.900.258.60

0.0250.83

2.00100.00

0.500.500.10

Enshi

944.00

492.00

808.00152.00

0.1219.52.822.020.210.22

Media mg L-1

APPENDIX I

Plant tissue culture media:WPM (McCown Woody Plant Medium, Lloyd and McCown 1981); DKW/Juglans medium (Driver and Kuniyuki 1984); MS medium (Murashige and Skoog 1962); Enshi medium (cited in Kozai et al. 1988, formula supplied by Kozai).

52

Preece, J. E., Christ, P. H., Ensenberger, L. andZhao, J. 1987. Micropropagation of ash(Fraxinus). Combined Proceedings ofInternational Plant Propagators Society 37: 366-372.

SAS. 1985. SAS Users Guide. SAS Institute,Inc., Cary, N.C., U.S.

Silveria, C. E. and Cottignies, A. 1993. Period ofharvest, sprouting ability of cuttings, and in vitroplant regeneration in Fraxinus excelsior. Can. J.Bot. 72: 261-267.

Tabrett, A. M. and Hammatt, N. 1992.Regeneration of shoots from embryo hypocotylsof common ash (Fraxinus excelsior L.) Plant CellReports 11: 514-518.

Young, J. A. and Young, C. G. 1992. Seeds ofwoody plants in North America. Revised andenlarged edition. Dioscorides Press, Portland,Oregon.

54

APPENDIX II

Abbreviations

BAP Benzyl aminio purineCDE Carbon dioxide enrichmentDKW DKW medium Driver and KuniyukiEC electrical conductivityGA

3Gibberellic acid

IAA Indole acetic acidIBA Indole butyric acidK KinetinMS Murashige and Skoog mediumNAA Napthalene acetic acidPAM Photoautotrophic micropropagationPGR Plant growth regulatorPPFD Photosynthetic photon flux densityTDZ ThidiazuronWPM Woody plant medium