"~

THE GROHTH AND DEVELOPMENTAL PHYSIOLOGY OF EUCA~ ._-.--::_Ix ,~"~"'" Ao

IN CELL AND TISSUE CULTURE SYST~I;~;~.· ~

{1!Jj;: <~:teca .

~', . . ,." (' , ",c3r'~ <.!;( ~..~ ,\~:v

•• ••••••-- ••• E Ali ••

Presented in Partia1 Fu1fi11ment of the Requirementsfor the Degree Master of Science

The Ohio State University1975

"tt1~~-- AdviserDepartment of Microbio1ogy

without whose help and support, the two years of study, research andfinal submission of this thesis would not have been possible.

the necessary financial support and in preparing for the trip to theUnited States, the author wishes to thank the administration of Escola

States Agency for International Development and the Fundacao de Amparoa Pesquisa de Sao Paulo. The author also acknowledges the continualassistance and advice provided by the Office of International Affairs ofthe College of Agriculture, The Ohio State University.

,Ran-ir Sondhal, David Evans and many others. This daily assistance was

Without the use of the greenhouse facilities, the research wouldnot have been possible. The author wishes to thank Or. Elton F. Paddock,Oepartment of Genetics, for this important contribution.

The author wishes to thank Adrienne M. van Zwoll for her inval-uable assistance in photographing research results in the laboratory.

Special thanks are due Or. William R. Sharp, adviser to theauthor, for his many hours of advice and assistance during the two yearstay at The Ohio State University in the Oepartment of Microbiology.

The author wishes to especially acknowledge the assistance andsupport of his wife, Randi Young Goncalves and her family for theirunfailing efforts and continual moral support during the difficultperiod of evaluating laboratory results, tabulation of data and finalsubmission of the thesis. The author specifically wishes to thank hisfather-in-law, Assistant Professor Clair W. Young, for his invaluableassistance in typing and editing the thesis.

Page

ACKNDWLEOGMENTS . ii

LIST DF FIGURES v

LIST DF TABLES viii

I. INTROOUCTION 1

lI. LITERATURE REVIEW 3

III. MATERIAL ANO METHOOS 98IV. RESULTS ANO OISCUSSION 116

V. CDNCLUSION -:-. H~2

VI. SUMMARY 184

BIBLIOGRAPHY 189

1. Main pheno1ic components produced in acid treatment ofEuca1yptus 1eaves (Hi11is, 1966a) . . . . . . . . . . . 21

2. F10w sheet summarization of the inter-re1ationship betweendifferent 1ines of experimentation opened by techniques ofp1ant tissue and ce11 cu1ture (Street, 1973) 32

3. Examp1es of commQU auxins, inc1uding some indo1e acids,naphta1ene acids, phenoxy acids and benzoic acids (Leopo1dand Kriedemann, 1975) . . . . . . . . . . . .. .. .. 45

4. Natura11y occurring cofactors and inhibitors of IAA oxidase(Leopo1d and Kriedemann, 1975). . . . . . . . . 48

5. Structures of some natura11y occurring synthetic purinecytokinin (Leopo1d and Kriedemann, 1975) .. . . . 51

6. The structures of some p1ant gibbere11ins and suggestedinterconversions between them (Leopo1d and Kriedemann,1975) . . . . . . . . . . . . .. . . . . 55

8. Some representative pheno1ic inhibitors of the f1avinium,cha1cone and depside categories. Common locations ofglucoside attachments are indicated by the G pointers(Leopo1d and Kriedemann, 1975) . .. 62

9. Some representative pheno1ic inhibitors. The origin frompheny1a1anine and tyrosine is indicated at the 1eft(Leopo1d and Kriedemann, 1975) .. . . 64

10. Some other types of growth inhibitors (Leopo1d andKriedemann, 1975) 67

11. Root morphogenesis in ca11us of Euca1yptus grandis 1eaf-b1ade exp1ant cu1tured in the Nash and Davies (1972)medium supp1ied with 40 mgj1 of sucrose . . . . . . . . .. 124

12. Ca11us growth habit of Euca1yptus randis 1eafb1adeexp1ants in the Nash and Davies (1972 medium with:

(a) 1.6-41.2 mg/l, and (b) 102.4 mg/l of H3B03 concentra-tions. Callus grown under 12 hour photoperioa . . . . . 128

13. Roots from explants of leafblade of adult Eucaly tusgrandis cultured in the Nash and Oavies (1972 medium,supplied with 3.2, 6.4, and 25.6 mg/l of H3B03. Culturegrown under 12 hour photoperiod . . . . . . . . . 130

14 Callus from leafblade explants of adult Eucalyptus grandisat the 60th day of culture in 12 hour photoperiod usingdifferent nitrogen sources and concentrations . . . . .. 134

15 Root development in explants of leafblades of adult Eucalyp-tus grandis in the Nash and Oavies (1972) medium suppliedsupplied with 1.0 mg/l of IBA and 0.0 mg/l of kinetin.Cultured in 12 hour photoperiod . . . . . . . . . . . . .. 138

16. Callus from explants of leafblade of adult Eucalyptusgrandis in the Nash and Oavies (1972) medium with 1.0 mg/lof kinetin plus 0.1 mg/l of 2,4-0. Culture grown inda rk n e s s _. • • • • • • • • • • • • • • • • • • • • • • • 146

17. Callus from Nash and Oavies (1972) medium subcultured inthe Sommer et al., (1975) No. 1 medium in the dark afterthe 8th weer-o~culture . . . . . . . . . . . . . . .. 150

18. Roots and shoots (arrows) morphogenesis in explant of leaf-blade of adult Eucaly~tus robusta with GA~ pre-treatmentand cultured in the ~ãsh and Oavies (1972) medium with1.0 mg/l IBA and 0.0 kinetin. Cultures grown under 12 hourphotoperiod 153

19. Callus produced from explant of leafblade of adult Eucalyp-tus grandis cultured in the Nash and Oavies (1972) medi um,supplemented with 15% coconut milk plus 1.0 mg/l of 2,4-0 157Callus of Eucaly}tus grandis hypocotyl cultured in the Nashand Oavies (1972 medium under 12 hour photoperiod ..... 160Example of small globular culture in the platingexperiment . . . . . . . . . . . . . . . . . . .

22. Shoot development from the internode of E. cumaldulensis(adult trees) in the Nash and Oavies (1972) medium with IBA(1.0 mg/l) devoid of kinetin, without the GA3 pre-treatmentand grown in the dark .•................. 173

23. Deve10pment of naked bud of I. grandis juveni1e, withoutGA3 pre-treatment, cu1tured in the Nash and Davies (1972)medium with IBA (1.0 mg/1) devoid of kinetine under 1ightconditions . . . . . . . . . . . . . . . . . . . .. 175

24. Deve10pment of naked bud and roots of I. grandis adu1t, innada1 cu1ture in the Nash and Davies (1972) with IBA (1.0mg/1) and devoid of kinetin. Cu1ture grown under 1ightconditions.. . . . . . . . . . . . . . . . . . . . . . . 177

25. Deve10pment of naked and adventitious buds af E. robustain the Nash and Davies (1972) medium with IBA {1.0 mg/1)devoid of kinetin. Cu1ture grawn in the dark . . . . . 179

2. Tree species used in tissue cu1ture regeneration roots,shoots, p1ant1ets and asexua1 embryos .... 78

3. Organs or parts of organs used and their method ofpreparation . . . . . . . . . . . . . . . . . . . 100

4. Composition of Murashige and Skoog (1962) medium . 101

6. Composition of Linsmáier and Skoog (1965) medium 1037. Composition of Tu1ecke et ~., (1965) medium 1048. Composition of Nash and Davies (1972) medium 1059. Composition of Sommer et ~., (1975) medium No. 1 106

10. Resu1ts of C1orox treatment used in steri1ization ofEuca1yptus grandis 1eafb1ade exp1ants . . . . . . . 118

11. Fresh and dry weight (mg) and the ratio between fresh anddry weights of ca11uses from 1eafb1ade of Euca1yptusgrandis cu1tured in Nash and Davies (1972) medium withdifferent sucrose concentratiors under 12 hour photoperiod 122

12. Ca11us characteristics, fresh and dry weights (mg) ofca11uses from leafb1ade of explants of adu1t Eucalyptusgrandis cultured in Nash and Davies (1972) medium andsupp1ied with different H3B03 concentrations. Evaluationat the 60th day of cu1ture in 12 hour photoperiod . . . .. 126

13. Fresh and dry weights (mg) of ca11us from adu1t Euca1yptusgrandis 1eafb1ade exp1ants. Cu1tured in severa1 combina-tions of nitrate and ammonia supp1ied to the Nash and Davies(1972) medium. . . . . . . . . . . . . . . . . . . .. 133

14. Fresh and dry weights (mg) of ca11uses from explants of·leafb1ades of adult Eucalyptus grandis. Culture grown in 12hour photoperiod for 60 days. IAA and Kinetin . . . 136

15. Fresh and dry weights (mg) of calluses from explants of1eafblade of adult Eucalyptus grandis. Cultures grown in12 hour photoperiod. IBA and kinetin . . . . . . . 141

16. Fresh and dry weights (mg) of calluses from explants of1eafb1ade of adult Eucalyptus grandis. Cultures grown in12 hour photoperi od for 60 days. NAA and kinetin . . . .. 143

17. Fresh and dry weights (mg) of cultures of exp1ants of leaf-blades of Eucalyptus grandis growing in 12 hour photo-period .. . . . . . . . . . . . . . . . . . . . . . . . . 144

18. Fresh and dry weights (mg) of cultures of leafb1adeexplants of adult Eucalyptus grandis in Nash and Davies(1972) medium under different 1ight conditions . . . . . 148

19. Fresh and dry weights (mg) of callus from Eucalyptusgrandis leafblade exp1ants . . . . . . . . . . . . . . . 152

20. Fresh and dry weights (mg) of calluses from explants of1eafblade cultured in the Nash and Drvies (1972) mediumwith 1.0 mg/l of IBA and 0.0 kinetin . . . . . . . . . . 156

21. Fresh and dry weights (mg) of Eucalyptus grandishypocotyl culture with coconut milk under 12 hourphotoperiod . . . . . . . . . . . . . . . . . . 162

22. Characteristics of plating culture of Eucalyptus grandisseedling materials . . . . . . . . . . . . . . . . . . . 165

23. Characteristics of plating cu1tures of Eucalyptus grandis(1 year old) . . . . . . . . . . . . . . . . . . . . . .. 166

24. Characteristics of plating cultures of Eucalyptus grandis1 1/2 years old . . . . . . . . . . . . . . . . . . . . .. 167

25. Characteristics of plating cultures of Eucalyptus grandisadult (6 years) . 168

26. Results of nodal (stem segment) culture of Eucalyptusspecies in the Nash and Davies (1972) medium with IBA(1.0 mg/l) devoid of kinetin . . . . . . . . . . . . . 172

~Trees are mostly propbgated sexuallybecause the vegetative propagation of trees old enough to have demon-strated their superior characteristics is often difficult (Cameron,1968; Thulin and Faulds, 1968). Vegetative propogation, however, ispreferred because superior characteristics/are maintained better than bysexual propogation. It is advantageous, therefore, to look for newmethods of vegetative propogation to be used where conventional proce-dures are unsatisfactory (Bonga, 1974).

Over the past 20 years, considerable progress has been made inapplying tissue culture techniques in breeding programs of horticulturaland agricultural crops (Nickell and Torrey, 1969; Bajaj and Bopp, 1971;Hildebrandt, 1971). The potential of tissue culture is not limited tovegetative propogation. Embryo culture has been used to produce other-wise incompatible hybrids, (Bajaj and Bopp, 1971); haploid cultures haveresulted in the production of homozygous diploids (Vasil, 1972) somaticcell hybridization through protoplast fusion is being considered for thedevelopment of new cultures (Cocking, 1972), and meristem culture hasyielded virus free clones (Smith and Murashige, 1970; Bajaj and Bopp,1971; and Hildebrandt, 1971).

tissues to a stage 1arger than mere seed1ings are from both gymnospermsand angiosperms (dicots) genus (Bonga, 1974; Durzan and Campbe11, 1974).

By far the most effective method of 1arge sca1e vegetative pro-pogation has been ce11 suspension cu1ture. Vasi1 and Vasi1 (1970) foundup to 100,000 embryoids fo11owing p1anting on agar medium. There are,

/however, a number of prob1ems inherent in this technique. ln spite ofthese prob1ems, it may be eventua11y possib1e to propagate most species,

For most species, this may take many years to accomp1ish and othertechniques shou1d therefore be considered. The best a1ternate methodwhich does not have some of the potentia1 advantages of ce11 suspensioncu1ture but is 1ike1y to 1ead to vegetative propogation sooner and withgreater ease is the organ (bud or noda1) cu1ture technique (Bonga, 1974;Durzan and Campbe11, 1974).

Since the discovery of Eucalyptus in 1770, a great number ofclassification schemes have been proposed based on the characteristicsof leaves, bark, oils, buds, fruits, cotyledons, etc. The system inuse today, however, is based on the shape of the anthers and has beendeveloped gradually by a number of workers since 1886 when it was firstproposed. ./

The genus Eucalyptus belongs to thefamily Myrtaceae, whichcontains about 90 genera and over 3,000 species. The Myrtaceaeoriginally included a tribe, the Lecythidae, whichis now generallytreated as a separate family, the Lecythidaceae and the Barringtoniaceae.

Niedenzu, in Engler and Prantl (1893), divided the Myrtaceaeinto two subfamilies: the Myrtroideae with fleshy fruits and theLeptospermoideae with dry fruits. The Myrtroideae are practicallyconfined to the tropics and subtropics with twenty-five of the thirty-nine genera being restricted to central and tropical South America. Theother genera are found chiefly in the Pacific area, Malaysia, andtropical Asia, with two genera only extending to Madagascar and Africa.The Leptospermoideae, on the other hand, are restricted to the southwestPacific area, including New Zealand, Australia, New Caledonia, NewGuinea, Malaysia, Burna, Indo-China, and South China. The only exceptionin this distribution is the monotypic Tepualia, found in Chile.

The subfami1y Leptospermoideae is divided into two tribes, theLeptospermae with dry, dehiscent fruits (capsu1es) containing a few or

up to ten. The tribe Chamae1aucieae, comprising thirteen genera, andmany species of the Leptospermae, are confinedto Austra1ia, the majorityof the Chamae1aucieae occurring on1y in the southwest part of the

Six subtribes of the Leptospermae are recognized theBackhousiinae, Metrosideiinae, Euca1yptinae, Leptosperminae, Ca1othamni-nae, and Baeckeinae. A11 these subtribes reach their highest deve10pmentin Austra1ia with the Baekhousiinae (Backhousia; Osbornia) and theCa10thamininae (Ca1othamnus; Beaufoi1ia; Eremea; Phymatocorpus; Rege1ia)

\The Leptosperminae inc1udes the genus Me1a1euca which extendsfrom Austra1ia through Ma1aysia to southern China. The sixth subtribe,Euca1yptinae consists of two genera on1y~ Angophora and Euca1yptus. Inthe third genus, Euca1yptopsis, C.T. White shows simi1arities in woodstructure to the Euca1yptinae (Dadswe11 and Ing1e, 1951; Ing1e andDadswe11, 1953) but in its morpho1ogy White (1951) and Po11en Pike(1956) indicated that it is more correct1y p1aced in Metrosiderinae.

anatomica1 simi1arities to Euca1yptus and Angophora, but its ordercharacteristics render its p1acement in the Euca1yptinae. Thisc1assification is doubtfu1 and the true position is sti11 to be fixed.

Angophora and Euca1yptus on1y.The first recorded attempt to arrange the Euca1yptus into some

kind of system was in 1799, when Wi1dman, in his Species P1antarum, used

Euca1yptus into two groups; one group having a conica1 opercu1a and theother hemispherica1 opercu1a. Wi11denow was fo11owed by Augustin deCando11e, who in 1828 pub1ished a c1assification scheme in Prodromus

secondary character. It was rea1ized 1ater, ~owever, that a11 specieshave opposite leaves in the ear1y stages (juveni1e 1eaves) and thissystem of c1assification fe11 into disfavor, a1though in 1843 Wa1pus1n Repertorium botanices Systematicae published a series of descriptionof Euca1yptus species using Cando11els system as his basis for c1assifi-tion.

von Mue11er, who separated Euca1yptus into six groups based on trunkbark characteristics: smooth bark, ha1f bark, wrink1ed persistent bark,

separates closely related species and brings together species which arenot closely al1ied. These factors, together with the difficulty of

accurately classifying species exactly, considerably lessens the overallvalue of this scheme.

useful and practical diagnostic character.Bentham (1866) can be regarded as the founder of the modern

classification system, as it he who first used the shape of the antheras a character by means of which species could be separated into groups.Bentham established five groups which were reduced by von Mueller(1882), increased considerably by Maiden (1903-33), and finallyelaborated by Blakely (1934) into eight sections and eighteen subsections.Blakely's antheral classification is the one in use today, and althoughit is by no means faultless, it is the only complete scheme to be

I

tion by Bentham, and its elaboration by Maiden and Blakely, two signifi-cant contributions to the problem of classification were published in1898; one by Luehmann and the other by Tate, concerning the value of thefruit as a diagnostic character. In the scheme proposed by Tate,Eucalyptus trees were classified according to fruit characters alone,taking into account the shape, sculpture, valves seeds, etc. Due to thegreat variability encountered in Eucalyptus fruits, however, a classifi-cation based on this organ alone is bound to be imperfect, and Tate's

fruit characters are still one of the most important classificationfeatures used today.

A number of other contributions to the specia1 prob1ems posedby the deve10pment of a classification system were made at about thesame time. Maiden (1889a~ 1889b, 1891) suggested using the chemicalnature of the kino as an aid to diagnosis~ and McAlpine and Renfrey(1890) proposed the use of cross sections of the petiole to assist inspecies determination. Von Mueller (1879-84) suggested using thecoty1edons and juvenile leaves as a basis for classification, but thiswork was never completed. By far the most elaborate system (apart fromantheral classification) and one that had a very stimulating effect onthe chemistry of Eucalyptus and consequently their utilization, was thec1assification of Baker and Smith (1920). This was based on the typeof oil present in the leaves, following the discovery of "physiologicalforms" or chemical varieties of a single species.

1. Antheral ClassificationsBentham (1866) established his system bf classification on the

anthers because he found that the order characters of Eucalyptus variedso widely that a satisfactory system could not be evolved. However, hedid not consider that anther shape was an ideal character on which toconsider a systematic arrangement of the genus and mentioned the factthat the groups graded into one another through a series of traditionalforms. Although Bentham relied upon the shape and mode of dihiscence ofthe anthers in the first instance to divide the genus into groups, healso used the fruit, the inflorescence and the receptacle as a secondarycharacter for further subdivision.

ln 1882, Von Mueller condensed Bentham's scheme. Later, in1884, Von Mueller (1879-1884) divided one of his groups into two.Maiden (1903-33), with more material on which to work, elaboratedconsiderably on Bentham's scheme and introduced six major sections withseveral subsections. As with other workers before him, Maiden placedthem as "miscellaneous.1I Maiden's scheme was admittedly incomplete, andhe himself considered it to be only a basis for future work when moreadequate material might become available.

The anthera1 classification of Blakely (1934) is the mostelaborate and complex of al1 Euca1yptus classification systems, and isthe one in general use today. It is outlined as fo11ows:

A. Section Macrantheraea. Section Macrantherae

Subsections: l. cordiformes2. ovoideae3. 1ongiores'4. truncatae5. subtruncatae6. orbiculares7. ovulares8. tereticornes

b. Section Macrantherae (normales)B. Section RenantheroideaeC. Section Renantherae

a. Section Renantherae

Subsections: 1. cordatae2. papi1ionantherae

b. Section Renantherae (norma1es)Subsection: 1. brachyandrae

O. Section Porantheriodeaea. Section Porantheriodeae

Subsection: 1. ób1iquiantheraeb. Section Porantheriodeae (norma1es)

Subsection: 1. attenuataeE. Section Termina1aeF. Section Graci1esG. Section MicrantheraeH. Section P1atyantherae

Subsection: 1. emarginatae2. adenophorae3. pyrioformes

2.. Secondary C1assification FeaturesAnther characteristics are primary features used in separating

the Euca1ypts into a number of groups. As in most cases, these groupscontain a 1arge number of species. A further series of characteristicsare used to subdivide each group into a number of series and subseries,each of which contains species c1ose1y a11ied to one another.

Among the most important of these secondary characteristics arebark type, timber characteristics, number of pairs of opposite juveni1eleaves, nature of the inflorescence, fruit characters, seed morpho1ogy,

and cotyledon shape.The delimiting of species within the eucalypts is beset with

many difficulties~ not only those inherent with the genus itself~ buta1so from the manner in which many species were originally described.Inadequate descriptions of species published in obscure non-botanicaljournals or sometimes garden catalogues~ and descriptions fromsingle specimens or even fragments~ are but a few of the difficultiesconfronting the serious student of Eucalyptus.

By giving greater attention to other morphological characters~most of the anomalies in classification have been recognized an9discussed in Australia for many years although little has been published.With this difficult genus~ as many taxonomic criteria as possible areneeded for reliable classification~ and the dangers in using a singlecharacter on which to erect taxonomic groups are recognized. "It willbe necessary to consider and evaluate all the evidence affecting eachparticular judgment and to distinguish between correiations of derivedcharacteristics which are likely to be highly significant indicationsof evolutionary relationships~ and unspecialized conditions which do nonecessarily suggest close affinity in the same way" (Pryor and Johnson(1962) in Hillis~ 1966 b).

In recent years, increasing attention has been given to woodanatomy~ bark anatomy, cytological studies~ pollen grains, seed coatanatomy~ floral morphology and polyphends in the leaves (Hillis~ 1966 aand b).

3. Euca1yptus Morpho1ogy and Anatomya. The Stem

Euca1yptus has tremendous variation in size at maturity,perhaps the greatest in the plant kingdom. They v~ry from smal1

100 minto the air, their straight trunks branch1ess for over 30 m ....:lI" JThe smallest Eucalyptus belong to those regions with poor soi1s and acid

climates, and particu1arly to those areas bordering the very drycentral regions of Austra1ia, which support on1y a stunted vegetation.These species are the smallest Euca1yptus with heights of on1y 2 mwhen ful1y grown. Upon removal to somewhat better conditions they maygrow a litt1e taller, but in their native habitat, they are mere1ydwarf shrubs. /

A wide range of shrubs and trees of various heights separatesthe very small Eucalyptus from the handfull of immense species. Thegiant trees occur in the well-watered mountains of the Southwest coast

Eucalyptus trees pass through several readily recognized stagesof growth and these stages have been fu11y described by Jacobs (1955)

and recognized two distinct groups of Euca1yptus separated by vesse1arrangement and distribution of the wood parenchyma.

Extensive studies on wood chemistry are reported by Penfo1d andWi1lis (1961). These authors reviewed the 1iterature where the wood

convenient for classificationpurposes. Oil glands are found in thebark of severa1 Euca1yptus trees. Comprehensive studies were carriedout by Chattaway (1955) and Carr and Carr (1969). The glands originate

The 1eaves of Eucalyptus show two very striking characteristics.Firstly, each individual tree deve10ps different kiyds of leaves atdifferent stages of its 1ife cycle with the two most familiar typesbeing the juvenile and adult leaves. Secondly, the 1eaf crown of the

exp~ains the outstanding vigor of Eucalyptus, particularly in countrieswhere they are not subject to the ravages of leaf eating insects. Theability to build a crown so quickly is due to the naked buds whichenable the tree to develop a number of branch orders in the space of afew weeks. Even if the crown is partially or wholly destroyed, the

All Eucalyptus, with few excepti ons, deve10p five differenttypes of leaves during their lifetime, each corresponding to certain

stages in the development of the tree. In order of development, theleaf types are: the cotyledons, tree seedling leaves, the juvenileleaves and the adult leaves. This phenomenon of heterophylly, whilepresent in most Eucalyptus, varies greatly in degree between differentspecies. In some cases, certain leaf types are not present at all, andif present, do not show the same striking dissimilarities of themajority species (Penfold and Willis, 1961). The shape of the 1eavesvaries from linear to cordate and its insertion can be: sessile,opposite and decussate, peltate, decurrent, amplexicaul and connate(Penfold and Willis, 1961).

The vast majority of Eucalyptus are evergreen( with the onlydeciduous members being a few tropical species which shed their leavesduring the summer months. However, for evergreen trees the life of theleaves is usually short, with Jacobs (1936) having shown that the averageof the leaves naturally occurring in Eucalyptus in about eighteenmonths. This of course, is subject to considerable variation with theperiod that the leaves remain on the tree depending on such factors asthe species concerned, the position on the tree, flowering and fruiting,the extent of insect attack and periodical bursts of growth. The rangeof variation is quite wide, with some 1eaves lasting for only a fewmonths while others may persist for four years or even longer.

In the axil of every Eucalyptus leaf is a stalked bud whichbecomes visible immediately when the subtending leaf unfolds. Thesebuds are known as naked buds, not being covered by protective scales as

gives rise to further shoots which carry on the growth of the tree.This process appears to be able to continue indefinitely (Penfold andWillis, 1961; and Cremer, 1972).

adventitious in origino As the stems of the Eucalyptus grow in diameterand the leaves are shed, the accessory pad of tissue accompanying each

ar three shafts of tissue which grow outwards, keeping pace with theradical growth of the stem and in the live bark or at the wood surface(Jacobs, 1955). If a cluster of shoots is formed from an epicormic bud,the basal ends fuse together to form a bulbous structure which Jacobs(1955) has termed the lIepicormic knob.1I

d. LignotubersMost species of Eucalyptus qevelop an underground swelling known

as a lignotuber, which is an organ of food storage and regeneration.

first, it takes the form of a pair of small axillary protuberances orswellings which develop at varying times after germination and which

lignotuber and can produce a juvenile leafy shoot, providing certainconditions are fulfilled. Some of the most important commercialEucalyptus do not develop lignotubers, however, a carrot-like swellingat the top of the root is found in other varieties and these swellingsact in a similar manner to the lignotuber but are readily killed byfire. Studies on lignotuber development and properties were made byChattaway (1958); Pryor (1957); Harris (1955); Jacobs (1955) and Blake(1972) .

applications. Brazil, with a wide variation of climate, has today about"110 species representing the largest plantation of Eucalyptus for

commercial application with uses mainly for paper and pulp (Andrade,

The Eucalyptus produce some of the heaviest, hardest, and mostdurable woods known. The quality of the timber, coupled with the rapidgrowth rate, regenerative powers and often great size, makes this genusthe most valuable source of hardwood in the world. However, only aboutsixty species, or approximately 10 per cent of the total species andvarieties known are important sources of timber at present. Many ofthe remaining species produce useful timber, but by reason of theircomparative smallness or rarity, they have not yet become economicallyimportant.

Eucalyptus species have been used as ornamentals, shade, andshelter trees in temperate or tropical climate countries. Since theearly nineteenth century, Eucalyptus, trees have been utilized foressential or volatile oils from their leaves, bark, buds flowers andyoung fruits. These oils have medicinal, industrial and perfume value.An additional use of Eucalyptus is that of honey flora.

In addition to the uses already described such as timber,essential oils, etc, Eucalyptus has the following applications: paperand 'pulp, fiber board, methyl alcohol, acetic acid, cellulose for themanufacture of rayon and celophone, ~oncentrated tannin extracts, andfuel such as charcoal for pig iron production. The leaves are used asfodder for various animals as well as being an important source of thedrug rutin. The roots of desert mallees provide certain native tribeswith water while the roots, seeds and nectar of some species are used bynatives as a source of food. Tannins for leather production are extractedfrom bark and kinos for pharmaceutical purposes are obtained as resinous

exudations on the bark, or as liquids from veins and pockets in thetimber (Penfold and Willis, 1961).

5. Eucalyptus Chemistry - PhenolsPhysiological form or chemical variety are terms used to define

forms or varities of species of plants indistinguishable from oneanother on morphological grounds, but which yield essential oils orother compounds of diverse chemical composition. This terminology hasbeen used in Eucalyptus taxonomy since the early 1920's (Penfold andWillis,1961).

In Eucalyptus, morphological leaf characteristics have beenused in determining the specific essential oils and their yield. Forexample, it was known that an obtuse venation of the leaf was indicativeof a low yield of oil, with pinene as the principal constituent. Amore acute venation with a marginal vein represented a slightly higheryield with cineole and pinene as the main constituents, while a"butterfly wingll venation indicated a very high yield with phellandreneand piperitone as the principal constituents (Penfold and Willis, 1961).

Variable compositions of polyphenols are found in the differentorgans of a tree. It depends on the stage of growth and development ofthe organs and the seasonal variations (Hillis, 1966a).

With the intensification of studies of chemataxonomy in thefamily Myrtaceae (Gibbs, 1974) and of the genus Eucalyptus (Hillis,1966 a and b); and of a better commercial use of the essential oils andpo1yphenols of Eucalyptus (Penfold and Willis, 1961; Hillis and Morita,

1969; Hillis and Ishikura, 1969; Bick et El., 1972; Hillis and Yazaky,1973 a), much of the polyphenolic composition of this genus has been

In I. dives, it was found that there is a significant differencebetween the mean oil yields of young and mature leaves and that young1eaves have a significantly lower piperitone content that do mature1eaves (Penfold and Willis, 1961).

Pryor and Willis (1954) obtained different compounds and yieldsfrom juvenile and adult leaves of E. aromaphleoia.

Hillis (1966b) studied fully grown juvenile and adult leaves ofE. accedens, I. dives, and I. ligustrina and found that juvenile leavesof these species contain significantly greater amounts of myricetin and

biosynthetic pathway. It is possible for samples of one species tocontain representatives of different classes of:

1. Non-convertible compounds, e.g. flavonoids-stilbenes; dihydro-chalcones-hydroxyxanthanes; phenylpropane (safrole)-monoterpenes(borneal).

2. Structurally related and possible interconvertible compounds,e.g. phellandrene-piperitone and possible also cineoli; limoene-pinene; scopolamine-hyoscyamine; angolensin-homopterocarpin;dihydrokaempferol-kaempferol.

Hattori et ~., (1954)Davies (1961)Penfo1d and Wi11is (1961)

Hi11is and Ishikura (1969)Bick et ~., (1972)

Nicho11s et ~., (1972)Hi11is and Yazaki (1973 a and b)

isovaleric aldehyde, cumina1, phe1-1andral, cryptone, terpenes,sesquiterpenes, cineole, amy1 andethy1 a1choho1s, isopropy1-pheno1,phe11andrene, cymene, pinene, gerany1acetate, geranio1 citrone1lal,eudesmo1, acetic acid, formic acid,ph1oroacetophene dimethy1 ether,carene, borneo1, nerolido1, rutin,tannins and f1avono1s.de1phinidin, cyanidin, pe1argonidin,myricetin, quercetin, kaempfero1,e11agic acid, caffeic acid, gentisicacid, ga11ic acid, p-coumaric acid,sinapic acid, feru1ic acid, macran-therin, renantherin, taxifo1in,aromadendrin, astringin, rhapontin,piceid, ch1orogenic acid,p-coumary1quimic acid and severa1unknownsrenantherin, macrantherin andhydroquinone

pinocembrin, a1pinetin and 0,0-dimethy1pinocembrincyc1oarteno1, sitostero1 and 24-methy1encyc1oartenol

methy1e11agic acids (16) and theirglycosides

inulin, raffinose, lyxose, prunasin,leucoanthyocyanins, syringin,skimic acid, quinic acid, l-quercitolsaponins, phenclic acids, malvidinglycosides, coumarins, phenolicesters and ethers and ketones.

Figure 1: Main phenolic components produced on acid treatment ofEuca1yptus 1eaves (Hi11is, 1966 a).

H0'Ç00:có"",R_'II , OHI T _

~ # OH R,HORI> R,=H; PelargonidinRI=OH, R,=H; CyanidinRI> Re=OH; Delphinidin

R I. Rc = H; K a~mpferolR,=OH, R,=H; QuercetinR" R,=OH; Myncetin

RI

HO-)-\;CH=CH-COOR,\.==/ ./R,

HO\.

RI·-!~· -\-COOH\=.1/ \

RI ·R,RI=OH, R2=H; GalJicacidR2 = OH, R J = H; Gentisic acid

RIReR,= H; p-Coumaric acidR IRe = H, R, = quinic acid; p-Coumarylquinic acidR, =OH, ReR3=H; Caffeic acidR,=OH, Re=H, R,=quinic acid; ChJorogenicacidR I= OCH3, R,R,= H; Ferulic acidRIR2=OCH3, RJ=H; Sinapicacid

R

HO~O,-, ~\-OHU j\=/~~! ~OH

, I

HO O

R=H; AromadendrinR=OH; Taxifolin

RI = H, R, =OH; PiceidR IR2 = OH; AstringinRI=OH. Rc=OCH3; Rhapontin

3. Compounds whieh differ only in the number of free hydroeylgroups, ete., e.g., kaempferol-quereetin-myrieetin; safrole-eugenol-methyleugenol-myristiein-elmiein; resveratol-rhaponti-genin; monoerotaline-speetabiline, sesamin-pinoresinol dimethylether.

4. Compounds whieh differ only in the position and type of sugarvariety or in their stereoehemistry, e.g., quereitin-isoquercitin-rutin; cateehin-epieateehin.

The following speeies were raised sueeessfully from euttings:Eucalyptus blakellyi, I. bieostata, I. globulus, I. maeulosa, I. grandis,I. bósistoama, and I. raeemosa. The basie eonditions neeessary forsuccess in raising Euealyptus from euttings were found to be: (1) theuse of juvenile fo1iage, (2) partia1 shade, and (3) the setting of thecutting in autumn or winter (Anonymous, 1948).

Fielding (1948) eonsiders that the sueeessfu1 striking ofEucalyptus euttings depend on two faetors: (1) the type of fo1iage used,and (2) the temperature and moist eonditions. Complete fai1ure wasexperieneed using euttings bearing adult 1eaves and the on1y sueeesswhich was obtained was from using shoots bearing juvenile 1eaves of thefullowing speeies: I. bieostata, I. blake11yi, I. dalrympleana,E. globu1us, I, grandis, I. maeu1osa, I. vinima1is, I. dives,E. robertsonii (I. radiata), I. bosistoana, E. bridgesiana, E. maero-rhyncha, and E. erebra.

I. robusta was sueeessfu11y propogated by euttings from youngtrees. SUrviva1 was better from 30 em than from 15 em euttings, but

with Na-a-haphthalene acetate gave positive results (Yoshisuji et ~.,1954) .

Claudot (1956) reported I. rostrata and I. viminalis vegetativepropogation from immature cuttings, air-layering, and grafting, but notgiving clear results.

Pryor (1957) found that it was much easier to graft shootsbearing juvenile leaves on to seedling stock. In some cases it had beenfound possible to graft one species on to another; for example,E. crebra on to E. ovata. For Pryor (1957), perhaps the most promisingmethod was the lIaerial-layeringll technique used with I. linearis.

Gurgel Filho (1959), studying the vegetative propogation ofseveral tropical forest trees, among them several Eucalyptus species,found that in general budding was more successful than grafting. Thiswas especially true when carried out in August or October. Layering wasnot successful for Eucalyptus and experiments with cuttings gave nopositive results.

Giordano (1960, 1960a) describes experiments propogatingEucalyptus by layering and by cuttings. Satisfactory results wereobtained by layering two year old I. rostrata. Experiments with cuttingsof a number of species confirmed the decisive importance of age ofparent. Rootings of cuttings taken from one year old plants dependlargely on the date of collection and the moisture of the medium.

Ruggeri (1960), in view of the extreme difficulty found inrooting cuttings of eucalypts, developed a study on the process of rootformation in E. rostrata, a species with which some success had beenobtained.

Giordano (1961) reported experiments made with f. botryoides andE. rostrata cuttings in sand or "agrilit" under continuous mistoE. botryoides all rooted after twenty days and E. rostrata cuttings fromone year old plants rooted satisfactorily, but those taken from adultplants did not root.

Penfold and Willis (1961) indicated that some success had beenobtained in Russia with E. rudis, E. cinerae, E. tereticornis andE. urnigera.

Cleft grafting of Eucalyptus scions from mature trees on toseedlings of the same species were developed in the open nursery in aheated greenhouse during winter with 80-100 per cent success of allspecies tried. Those species involved were: f. saligna, f. botryoides,I. malleriana, E. ficifolia, and E. leucoxylon (Thulin and Faulds,1962) •

Irrigation with complete nutrient solution and nitrogen compoundsalone or together with sucrose appeared to play some role in the 1eafeffects in the treatment with kinetin on rooting of E. cama1dulensiscuttings (Bache1ard and Stowe, 1963).

Marcavi11aca and Montaldi (1963) tested various methods ofvegetative propogation with f. rostrata. The most success (70 per centrooting) was obtained with vegetative shoots from stoo1s cut back to oneto five meters above ground 1eve1. These cuttings were taken in May andJune. heated with IVA, and p1aced in a 1-1 mixture of sand and trituredlava. Layering (both ground and air) of shoots from ten-year trees wasunsuccessfu1 and cuttings from the upper parts of these fai1ed to root.

A few successes in 1ayering were, however, obtained with shoots of two-year trees.

Franc1et (1963), in an account of experiments in Tunisia inpropogation by cuttings, stated that species cou1d be successfu11ypropogated in sand with mist irrigation and treatment with growthfactors: I. rostrata (90-100%), I. gamphocepha1a (90-100%),E. occidenta1is (100%), I. cneorofo1ia (86%), I. diversico1ar (82%),f· odorata (82%), I· p1atypus (76%), I. pi1iguensis (62%), I. bicostata(62%), and I. vinima1is (60%).

Pryor and Wi11ing (1963), in an account of progress, reportedthat vegetative propogation of Euca1yptus had been achieved mainly bygrafting an air-1ayering had a1so been very successfu1. They found thatcuttings wi11 root if sections cut from stems of very young seed1ingsare used.

Ruggeri (1963) using I. cama1du1ensis, made cuttings from dif-ferent 1eve1s in the stem of one-year seed1ings and from mature p1antsused under mist in two media; sand and "agrilit'" Root formationoccurred on1y in the young material and a1ways originated in the cambium,often accompanied by ca11us formation, the degree of which differed inbasal and apica1 cuttings. Rooting appeared to be re1ated primari1y toage of cutting, but was a1so affected by po1arity, rooting medi um, andmist treatment.

Boden (1964) studied the deve10pment of desirab1e rootstocks andthe behavior of graftings between I. camphora (which occurred on heavysoilswith impeded drainage) and E. da1rymp1eana (found in and adjacent

but we11 drained site). The resu1ts indicated that there was surviva1of grafts in both sites.

Marcavi11aca and Monta1di (1964) reviewed work from 1938 to 1961in the rooting of I. rostrata and tabulated results of local experimentstesting air and subterranean l~ers and cuttings and leaf cuttings fromwood and plants of various ages taken at various seasons and treatedwith: (1) growth regulators, (2) different mechanical treatments in thecuttings, and (3) different rooting media. The success of methods didnotadd new facts to the known difficulties in the vegetative propogationof adult trees.

Fazio (1964) propogating I. rostrata and f. polyanthemos bycuttings from trees older than ten years (other than basal shoots) andt~es with iron chlorosis did not experience successful rooting.

Ruggeri (1966) made anatomical observations on cuttings of~. carnaldu1ensi s (f. rostrata) propogated by hydrophoni cs using runni ng~ter and six-month seedlings and two-year p1ants of shoots. Changesintissues directly affected by the cut, increasing with age of shoot,werefound in shoots of two-year plants which showed abundant callusformationand rooted with difficulty, however, material of seedlingsrootedrapidly in ten to fifteen days.

Franclet (1970), outlining the objectives of research inTunisiaon the propogation of E. cama1du1ensis by cutting, explained areliab1etechnique for the satistical interpretation of experimentoFranc1etdescribes studies, buth on factors inherent in the propogating~terial itself and external factors believed to influence rooting. Theexperiments showed that under Tuni sian conditi ons, mass producti on of

o camaldulensis by cuttings from young plants is practical but problemsof propogation from older trees remain to be solved.

Paton et ~., (1970) through available physiological evidence,suggests that ontogenetic ageing of f. grandis seedlings involves adirect and quantative association between decreased rooting ability ofstem cuttings and increased levels of a rooting inhibitor in the tissueforming the base of the cutting. As detected by bioassay, thisinhibitor is present only in adult tissue which very rarely roots fromstem cuttings. It is absent in easily rooted seedling stems of allEucalyptus species tested but it is also absent in the easily rootedadult tissue of the exceptional species of f. deglupta.

In contrast to these statements, Davidson (1974) studied anddescribed effects on rooting ability of cuttings of f. deglupta, the~sition of the seedling stem, and the-ontogenetic age. In cuttingstakenfrom three month old seedling position, the cuttings on the shootsystemhad no effect on rooting or subsequent growth. Cuttings rootedfreelywhen taken from upper parts of trees up to one year old and theyappearedto grow exactly like seedlings. Corresponding material fromtreesaged five years and more completely failed to root. Stem cuttingsofE. deglupta were used as bioassay materials to test extracts fromtissueof various ontogenetic ages. Responses were clear-cut andindicatethat the failure of cuttings from older trees was due torootinginhibitors. A technique of rooting large numbers of cuttings~s described. Almost 100% success in rooting was achieved after eight~eks in mist propogation.

Dutt et ~., (l971) succeeded in inducing rooting of simpleair-layers in f. sieberiana, and simple-layer in E. macarthuri. Thesimple-layers in I. sieberiana took almost seven months to root whereasthe air-layers rooted within 2 to 4 months of girdling. The simple-layers in E. macarthuri rooted in about three months time.

Blake (1972) studied the effects of seasonal and nutritionalfactors on dormant bud development on the lignotubers of f. obliqua.Seasonal variations in apical dominance, maximal in summer and minimalin winter, were noted in lignotubers. Temperature variations were moreimportant than photoperiod in affecting seasonal variations of apicaldominance. The concentrations of growth promoting and growth inhibitingsubstances in the leaves were coorelated with seasonal variationssuggesting that both have a role in apical dominance. Decapitationstudies indicated that amounts of mineral nutrients and carbohydrates donot prevent dormant bud development.

Burgess (1974), studying grafting of f. grandis, was notsuccessful in Northern New Wales because of delayed incompatabi1ity.Attempts to root cuttings from seedlings and coppice shoots from thebases of older trees were moderately successfu1.

Davidson (1974 a) applied several methods of grafting toE. deg1upta and repeated fai1ure of the stock occurred in methods otherthan patch grafting. It was postu1ated that an inhibitor of the stockwas caused by adult scion material. Lines of further research onincompatabi1ity were suggested.

Suiter Filho and Yonezawa (1974) reported that scions fromselected trees of ~. saligna differed in survival following grafting.method was the side graft and the least effective was budding. Boththe cleft and splice methods of grafting provided some degree ofsuccess.

1. General Tissue CultureWhen Haberlandt attempted the first plant cell culture, his

intentions were to develop a more versatile tool to explore morpho-genesis and to demonstrate totipotentiality of plant cells. He probablydid not suspect that the cell culture technique would become a valuableaid in economically oriented activities. There are four areas inwhich applications of plant tissue culture are possible, either presentlyor in the near future: (1) production of pharmaceuticals and othernatural products, (2) the genetic improvement of crops, (3) the recoveryof disease-free clones and preservation of valuable germ plasma, and(4) rapid clonel multiplication of selected varieties. Although therehas been substantial research on the use of cell and organ cultures assources of pharmaceuticals, a successful commercial application remainsunrealized.

The technique of embryo culture has been used by plant hybridizersfor many years. Test tube pollination with ovary and ovule cultures maysimilarly find utilization in plant breeding. Two recent developmentsin cell culture techniques have been stirring considerable excitement.One has been the attainment of haploid plants through anther and

microspore culture. Haploid plants will minimize hybridization stepsconsiderably and help markedly in the recovery of mutants, eithernaturally or artificially induced. The other development has been thefusion of protoplast of genetica11y distinct species and the achievementof somatic hybrid p1ants.

Plant researchers may soon expect to achieve genetic transforma-tion of higher p1ants. It is conceivable that ultimately an alternateand much abbreviated procedure for developing a new crop variety wil1involve the introduction of desired cistron units from one variety intothe protoplasts of another. This will be fo1lowed bythe reconstitutionof p1antlets from the transformed protoplast.

The use of cell and organ culture methods in the re-establishmentof pathogen-free clones has been reviewed recently. Murashige andJones (In press). These methods result in the restoration of vigor andyield lost due to infection. Furthermore, they provide a means toexpedite the movement of plants from one country ar region to another;they could greatly relieve quarantine pressures. If combined withfreeze preservation methods, ce1l and organ cultures will also providea method for the conservation of valuable pathogen-free germ plasma(Murashige, 1974).

In Figure 2, the various types of sterile culture are shownwithin the rectang1es. The interconversions possible or projected areshown by arrows, interruptéd as appropriate to name the processes invokedor treatments used. The production of new plants ar cell cu1turesinviting detai1ed biochemica1 and physio1ogical study are shown as end

Figure 2: F10w sheet summarization of the inter-re1ationship betweendifferent 1ines of experimentation opened by the techniquesof p1ant tissue and ce11 cu1ture (Street, 1973).

~ ANTHERS

CHOSEN PLANT SPECIESAND VARIETlES

(2n)

1VEGETATlVE

ORGANS

products of the different lines of experimentation (n = haploidchromosome number, 2n = diploid chromosome number).

The available information on tissue culture propogation ofplants has been obtained from experiments with herbaceous but notwoody genera. Accordingly, the conclusions of some research findingsshould not be viewed as applicable to the propogation of woody perren-ials, except perhaps as a foundation for their research. The successfulmanipulation of plant or organ regeneration processes has thus far beenconfined to those plants whose cuttings can be rooted without difficulty.Hence, even some tree species such as the aspen have been propogatedsuccessfully through tissue culture, although most have noto Areasonable guide is that any herbaceous plant whose cuttings can berooted easily is multiplicable through tissue culture following aminimum of experimentation.

The goal of asexual propogation is to produce uniform plants ofa selected genotype. Vegetative propogation usually assures that thedesired characteristics of the selected plant is retained throughout itsclone. In contrast, there is no certainty that seedlings will reproduceeven some of the parental characteristics. Nevertheless, seed propoga-tion has been used in many instances for economical reasons, and majorcharacteristics of a variety have been reproduced with sufficientconsistency, e.g., many flowers and vegetables.

It is possible to achieve diverse objectives through tissueculture methods of plant propogation. It is most often sought as analtern~tive in the propogation of cultivars when conventional methodspermit only slow increases in clonal plants, e.g., orchids. Tissue

culture propogation is particularly helpful when used in conjunctionwith plant breeding programs. It enables the timely increase and hastensthe ayai1ability of new varieties. Even with cultivars that arepropogated readi1y through cuttings, divisions, and other conventiona1asexual techniques, the tissue culture method can be utilized toenhance substantially the rate of multip1ication. A millionfold increaseper year in the rate of clonal multip1ication over conventiona1 methodsis not unrea1istic. Hence, with p1ants such as chrysanthemums andothers that are already propogated quite rapidly by stem cuttings, tissueculture may be a desirable aid in meeting special needs. In commercia1nurseries, tissue culture can a1so be used to minimize the growing spaceusually provided for the maintenance of stock p1ants. Furthermore, whenproperly executed, the method can also be used in the reproduction angmaintenance of relatively disease-free p1ants.

a. Selected Aspects of Media Compositioni. Inorganic and Nitrogen Nutrition

The inorganic salt solutions now used to supp1y mineral e1ementsto plant tissue cultures have been developed by modifying the solutionsused by Gautheret (1935 and 1937) and White (1943) in their pioneer workon the conditions necessary for the continuous growth in the culture oftissues of callus origin (Street and Henshaw, 1966). A number ofworkers demonstrated that the inorganic salt additions of White andGautheret were suboptional (Heller, 1953; Hildebrandt et ~., 1946 andMurashige and Skoog, 1962). Enhanced growth of a number of tissues wasobtained by using considerably enhanced levels of potassium and nitrogen

biosynthetic potentialities of the altered tissue is directed todifferences in physiology between normal, habituated, and crown galltissue cultures of the same plant origin (Street, 1969). In this area,the same problems that have beset those who interpret mineral nutritionof rooted plants also arise. Among these are the distinctions, not

cL,l~-always clear, between effects dur to total supply and concentration ofa solute in question and the way absorption may be affected by aeration

ÚI)Jr bobsorb them from great dilutions; nevertheless, the access of ions to

always easy to distinguish unequivocally, which has occurred or by whatfactors it may have been regulated (Steward, 1969).

tissue capable of active growth on a defined medium dispersed with theuse of agar (which is a source of calcium, magnesium, and a number ofmicroelements). The cultures were supported at the surface of the liquid

culture medium by ashless filter paper. Growth was enhanced if good

a fi1m of si1ica ge1. A modification of this technique, which permitsapparent1y by renewing the gas phase in the culture tubes (Heller, 1965).

Using·this 1iquid medium technique, He11er has demonstratedthat ca11us cu1tures require nitrogen, potassium, magnesium, phosphorous,

~ ~QOsu1phus, iron, zinc, boron, magnanese and copper. The question of

critica11y. Sodium on1y slight1y delays the death of tissues deprivedof potassium. The other estab1ished requirements were simi1ar1y shown

ii. Nitrogen NutritionRicker and Gutsche (1948), Nicke11 and Burkho1der (1950) and

He11er (1953) reported that nitrogen is required as nitrate and thattheir ca11us cu1tures cou1d not, even at neutra1 pH, utilize nitrite or

the uti1ization of ammonium by cu1tured roots, (Street, 1969), theconc1usions were not the same. Shantz and Steward (1959) found that

amino acids for the growth of exp1anted carrot tissue. Steinhart, et ~.,(1961) found that urea was an efficient source of nitrogen for spruce(Picea) tissue. The possibi1ity that the activity of urea as a nitrogen

ammonium has been raised (Shantz and Steward, 1959).

Riker and Gutsche (1948) found that the following single aminoacids, although inferior to nitrate, could support the growth of sun-flowers (Helianthus) normal and crown gall tissues: alanine, glycine,arginine, glutamic acid, aspartic acid, and asparagine. Nitsch andNitsch (1957) reported that callus cultures of Helianthus tuberosusgrew as well in alanine, y-aminobutyric acid, glutamic acid, asparticacid, glutamine, or urea as with nitrate during a 21 day growth period;and Demetriades (1958) found for several normal callus cultures thataspartic acid and sometimes glutamic acid or arginine could effectivelyreplace the nitrate requirements. It must, however, be emphasized thatfor many cultured tissues~ single amino acids or amino acid mixtures areeither completely ineffective as sole sources of nitrogen or are markedlyinferior to nitrate.

Any seeming ineffectiveness of amino acids cannot be due toinability of the cultured tissues to absorb amino acids, for whensupplied as 14C-labeled compounds, they are readily absorbed andmetabolized. The results suggest rather that the absorbed amino acidsmay not always be actively deaminated or that their deamination doesnotyield ammonium at sites where it can be effectively used for thesynthesis of such primary products of nitrate assimilation as glutamicacid, aspartic acid, alanine, and arginine are ineffective. Thesecells have an absolute requirement for nitrate because certain inter-mediates in nitrate reduction is involved in the biosynthesis of otheressential nitrogenous constituents.

Menoret and Morel (1958) concluded, from studies with carrotroot explants, that the conditions of tissue culture caused profound

changes in their free amino acid composition. The tissue at the time ofexcission from the carrot was rich in arginine, glutamine, asparagine,and glutamic acid. After a period in culture~ the tissue did notcontain a detectable level of arginine. The amounts of the amides andof glutamic acid had marked1y decreased re1ative to other amino acidsand the a1amine content had risen to account for 40% of the free aminonitrogen of the tissue. Duraton (1959) found that the tuber tissue ofHe1ianthus tuberosus was extreme1y rich in arginine but the amino acidcomposition changed very rapid1y when the tissue was excised and p1acedon culture medium. Arginine disappeared and the 1eve1s, particu1ar1y ofproline but a1so of hydroxy-L-pro1ine, glutamic acid, the amides, anda1anine also increased. This uti1ization of the store of arginine bytissue initiating growth of cu1ture fo11ows a different course thanthat of arginine uti1ization in the deve10ping shoot or seedling wherepro1ine does not accumu1ate and its carbon becomes incorporated in new1ysynthesized organic acids, sugar, and chlorophy11 pigments (Duraton andMai11e, 1961 a and 1962).

Amorin (1970) using Paul's Scar1et Rose tissue, reported thatthe increase of the 1eve1s of nitrate in the media decreased the produc-tion of pheno1ics.

iii. Carbon Nutrition and Metabo1ismThe carbohydrate requirement of a ca11us culture was first

studied experimenta11y by Gautheret (1941 and 1945), who examined theabi1ity of various sugars to support the growth of carrot root ca11us.Similar studies invo1ving normal and tumor tissues of a number of

dicotyledons were subsequently made by several researchers (Henderson,1954; Hildebrandt and Riker, 1949 and 1953; and Nickell and Burkholder,1950). Straus and LaRue (1954) studied the carbohydrate requirementof a culture derived from çorn endosperm, and Ball (1953 and 1955) of aSequoia callus. The above studies revealed that most cultures grow thebest when supplied with sucrose, glucose, or fructose. Galactose hasproved ineffective in most cases, although for normal and tumor tissueof Catharanthus roseus (Hildebrandt and Riker, 1949 and 1953) and normaltissue of Sequoia sempervirens (Ball, 1953) it is reported to be a verysatisfactory carbon source.

Callus cultures derived from different species or varietiesdiffer, often quite dramatically, in their ability to utilize differentcarbohydrates as their sole sources of carbon and energy. Suchcultures, therefore, may offer opportunity for comparative studies incarbohydrate metabolism, particularly through the use of 14C-labeledsugars. However, these potentialities have not been extensivelyexploited. Goris (1954) showed that carrot callus readily interconvertsglucose and fructose and synthesizes sucrose when supplied with eitherDf these monosaccarides. Similar synthesis and interconversion hasa1sobeen demonstrated in other callus tissues, and Bove et ~., (1957)have found that such sucrose synthesis in culture of Citrus limon ismarkedly reduced under conditions of potassium deficiency. Analyses ofculturemedia have shown that sugars synthesized in callus cells may bein part, releasedinto the surrounding medium (Barnoud, 1952). Doubtless,thisis partially a function of the activity in growth of the culture

and also of the number of the senescent cells it may contain (Street,1969).

The soluble carbohydrate composition of cultured tissues oftendiffers markedly from that of the plant tissues from which they arederived. This is also true of almost every class of metabolites. Thepresence or absence of added auxins or other growth regulators in themedium greatly influences the content of soluble sugars and othermetabolites (e.g., nitrogenous). Studies have been made of the influenceof auxins on the rate of sugar depletion in callus cultures transferredto sugar-free medium (Cova, 1943; Goris, 1947 and 1948 and a) and ofsugar accumulation in response to additions of auxins to sugar-containingmedia (Goris, 1950 and 1954). Such studies point to enhanced rates,both of uptake and of metabolic utilization of sugars, in response toauxins. There is evidence that 1ight, quite apart from its involvementin photosynthesis. promotes sugar uptake and metabolism in certaincu1tured tissues (Naef, 1959). Goris and Duhamet (1958) also found,in work with carrot cultures, that the sucrose level increased and there1ative level of reducing sugars corresponsingly decreased as the amountof coconut milk in the medium was progressive1y increased.

Amorin (1970), working with Paul's Scarlet Rose tissue, foundthat the increase in the sucrose 1evel in the medium had a correspondingincrease in the pheno1ic production by the cells.

iv. VitaminsCa1lus cultures, in contrast to root cu1tures and certain other

organ cultures, often do not require for growth the addition of recognized

vitamins to their cu1ture media. Pyridoxine (Wo1ter, 1964) and niacin·(Risser and White, 1964) rare1y seem to be essentia1, and in many cases,there is no convincing evidence that additions of these B vitaminssignificant1y enhance growth of Nicotiana tobacum (Linsmaier and Skoog,1965). The response of ca11us tissue derived from Nicotiana tobacumvariety IIWisconsin No. 3811 to thiamine is particu1ar1y interesting. At1east 400 ~g/l is required for maximum yie1d, and the yie1d increases1inear1y re1ative to the 10g of the concentration of thiamine over mostof the range 0.5-400 ~g/l. It has now been reported that this tissuecan grow slowly in the absence of an external supply of thiamine if themedium contains a high level (500-100 ~g/l) of kinetine (Digby andSkoog, 1966) and that this 1eve1 of kinetin activates thiamine synthesisin the cu1ture. Nicke11 (1952) reported that thiamine was essentia1 forthe cu1ture of wound virus tumor tissue of Rumex, but more recent1y thistissue has been described as thiamine autotrophic (Nicke11, 1961).Czosnowski (1952) demonstrated that the growth of certain ca11uscu1tures was not enhanced by externa1 thiamine as they contained thisvitamin.

Pantothenic acid is described as enhancing the growth of normalca11us of Crataegus (More1, 1946) and of crown ga11 tissue of Daturainnoxia (Schmidt and Brucker, 1959 and 1959 a).

Ascorbic acid enhances the growth of Picea glauca tissues(Reinert and Schraudo1f, 1959).

Fo11owing the iso1ation of the hexito1s from coconut mi1k, ithas been shown that externa11y supp1ied cyc1itols (particular1y myo-

inositol) are frequently stimulatory to the growth of callus culture(Linsmaier and Skogg, 1965; Narayana, 1963; Pol1ard et ~., 1961; Risserand White, 1964; and Steinhart et ~., 1962).

The need for other vitamins remains equivocal, although manyhave been included in the media (Murashige, 1974).

v. Growth Factors and InhibitersGrowth factors have been separated as a class of organic

compounds which influence the guality of growth in vitro more than thequantity. Thus, the category includes the natural hormones as well assynthetic hormones and growth regulators. These compounds are importantin the induction and maintenance of callus but also in the induction ofcel1 differentiation and morphogenesis. Since these compounds areproducts of cell metabolism, the possible endogenous production ofhormones must always be considered in the interpretation of experimentaltreatments. There are also mechanism for the destruction of hormoneswithin the cell (Sharp and Caldas, 1970). The major -9roups of hormonesare the auxins, the cytokinins, the gibberellins and the inhibitors. Ofthese, the first two classes (auxins and cytokinins) are the most widelyused in tissue culture because of their wide effectiveness in inducingcallus growth and morphogenesis. There are also numerous studies on theinteraction of these two types of hormones. There is relatively littleinformation about the effects of the other types of hormones in vitro.

The chief auxins used in tissue culture are indoleacetic acid(IAA), indolebutyric acid (IBA), naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-0). Only lAA is a natural auxin; it isvery sensitive to biological degradation unlike NAA, IBA and 2,4-0 which

are stab1e in solution. Oifferences exist in the effectiveness of theseauxins in ca11us induction for different species: species specificityfor NAA, lAA or 2,4-0 was seen in 12 species of conifers (Harvey andGrasham, 1969) and in Pinus, 2,4-0 is better than lAA for ca11us growth(Brown and Laurence, 1968). Other species in which 2,4-0 is better thanother auxins inc1ude rice, variety Kyoto which produces ca11us witheither 2,4-D or lAA, but rice variety Taichung deve10ps ca11us on1y whentreated with 2,4-0 (Yamada et ~., 1967; Wu and Li, 1971); Oioscorea(Rao, 1969); and 39 species of Nicotiana and 9 species of Popu1us(Matsumoto et ~., 1971). A1though 2,4-0 is genera11y the most effectiveauxin in ca11us induction, there are cases such as that of P1umbago, inwhich IAA is more successfu1 (Nitsch and Nitsch, 1967) or in whichanother auxin has given satisfacto!,y resu1ts (NAAinduces cal1us onros'eshoot tips, Jacobs et -ªl., 1969).

Examp1es of common auxins can be seen in Figure 3.Leshem (1973) discussed the mode of auxins action and out1ined

the possib1e medes as fo110ws: (a) transcriptiona1 regu1ation bymembrane-bound auxin acceptor protein, (b) auxin-RNA comp1ex, (c) auxincomp1exes with other nuc1eic components, '(d) effects on enzyme systemsassociated with ce11 wa11s, (e) auxins and the Go1gi apparatus, and(f) a110steric effect of auxin.

Antiauxins are chemica1s with c10se resemb1ance to auxins butlacking at 1east one requirement for activity (McRae and Bonner, 1953).The first report of a substance which cou1d 1imit the action of auxinswas that of y-pheny1butyric acid (Skoog et ~., 1942), and the antiauxinof trans-cinnamic acid was reported by Overbeek et ~., (1951). Some

Figure 3: Examp1es of common auxins, inc1uding some indo1e acids,naphtha1ene acids, phenoxy acids, and benzoic acids (Leopoldand Kriedemann, 1975).

~-- -------'-

OJCH'COOH OJCH'CH'COOH OJ CH,CH,CH,COOH~ N ~ N _

H H HIndoleacetic acid - Indolepropionic acid Indolebutyric acid

CH2COOH

O) COOCH,COOH~ fi ~ ~

Nophthaleneacetic acid ,I3-Naphthoxyocetic acid

OOCH,COOH OOCH,COOH CluO "H,COOHCl~ Cl ~ Cl Cl ~ Cl

4-Chloro 2,4 - Dichloro 2,4,5 -Trichlorophenoxyocetic acid phenoxyacetic acid phenoxyacetic acid

Cl Cl

oCOOH oC~OHNClÜCOOH

Cl ~ Cl ~ Cl Cl ClCl NH2

2,4,6- Trichloro 2.3,6-Tnchloro 4- Amino-3, 5,6 trichlorobenzoic ocid benloic acid picolinic acid

naturally occurring cofactors and inhibitors of IAA oxidase are shown inFigure 4.

Three major features of auxins stand out strikingly: (a) thediversity of auxins effects, (b) the diversity of the other chemicalcontrols which may be interwoven with the auxin effects, and (c) thesystemic patterns of the auxin effects. The diverse effects of ~uxinsare readily apparent in the large and expanding list of growth anddifferentiation activities which are influenced by endogenous auxins oraltered by exogenous auxins. In addition to cell elongation, apicaldominance, and abscission processes, there are effects on flowerinitiation and development, pollen tube growth, fruit set, fruit growth,and in addition, the formation of compression wood in conifers, tuberand bulb formation and seed germination. A1most every dynamic part ofthe plant growth and development seems to be affected by auxin. In theindividual cel1 there are effects on the plasticity and elasticity ofthe wal1, on cytoplasmic viscosity, on protop1asm streaming, onrespiration rates, on metabo1ic pathways, on changes in oxidativestates, on the contents of nucleic acids, and on the activities of manyenzymes. The auxin influences on physio10gica1 and developmentalprocesses general1y require other substances as cofactors (Leopo1d andKriedemann, 1975).

vi. CytokininsThe cytokinins are a group of growth substances which are

usual1y derivatives of the nucleic purine base adenine. However, othersubstances such as diphenyl urea or derivatives of nicotinamide are

'igure4: Natura11y occurring cofactors and inhibitors of IAA oxidase(Leopo1d and Kriedemann, 1975).

MHOV ·JOOH

HO~ >OH

~ Ó oHO 11 "{glucosel3

O "O~ I

HO~CH=CHCO

~CH20H

HOtJ-

HO~

HOV JOOH

OHHO~ >OH

~ Ó OHO 11 "{glucoselO ,,3

O~ I

HO~CH=CHCO

HOMHOV J - O---('j COOHHOV

OH

a1so ab1e to promote the process of ce11 division or cytokinesis (Woodet ~., 1969). The structures of some natura11y occurring and syntheticpurine cytokinins are presented in Figure 5.

Far from being ce11 division hormones in the strict sense,cytokinins participate in ce11 en1argement, tissue differentiation,corre1ation effects, dormancy, severa1 phases of f10wering and fruiting,and the regu1ation of senescence (Leopo1d and Kriedemann, 1975).

A11 cytokinin active forms are adenine derivatives with theside chain at the N6 position. Tests suggest that the imidazo1e ringis essentia1 for cytokinin activity. The optimum 1ength for theside chain is four to five carbon atoms. Doub1e bonds in the chainincrease activity. The re1ative activities conferred by ring substi-tuents are as follows: benzyl>fururyl = phenyl> themyl>cyclohexylpyrimidyl=pyrol=naphtyl-cyclopropyl. The planetary of the sidechain mayaffect the activity; there are differences~1n activity between the cisand trans forms (Skoog and Armstrong, 1970).

Cytokinin has been shown to influence specific e~yme activities.The rates of synthesis of certain enzymes in germinating rye appear tobe 1ncreased by cytokinins. The effect of cytokinin depends on thestage of deve10pment. This suggests that cytokinins are probab1y notinvolved in nuclear gene depression. The specificity of cytokinineffects on enzyme synthesis depends on the genetic programming of thecell rather than on the hormone itself (Skoog and Armstrong, 1970).

Since cytokinins occur in transfer RNA, they may control proteinsynthesis at the translationa1 level. Chemica1 modification of thecytokinin substituent results in greatly reduced binding to the

igure 5: Structures of some natura11y occurring synthetic purinecytokinin (Leopo1d and Kriedemann, 1975).

Q)=>N H

Adenine

/CH3H/CH2 CN , ~ 'CHo=:~ ,N H

Dimelhylallyladenine (DMAA)

ribosomes. Labe1ed cytokinin is incorporated into tRNA. However, minornitrogen bases such as cytokinin nuc1eotides are usua11y formed bymodification in the tRNA. There is no corre1ation between the degree ofcytokinin incorporated and cytokinin activity. Transfer RNA may be thesite of the cytokinin synthesis rather than its site of action (Audus,1972). Whi1e there is a possibi1ity that this hormone may act througha direct participation in nuc1eic acid functioning, aside from theoccurrence of cytokinin components in tRNAs, there is 1itt1e evidenceon which to base an exp1anation of its function through such a structura1invo1vement. As with auxin and gibbere11in, there are 1arge changes inthe nuc1eic acid and in protein synthesis and degradation associated withthe hormone action, but it becomes very risky to atrribute the changesin these synthetic systems to a direct action of the hormone. There isa doubt about where the actua1 site of hormone activity might be interms of intrace11u1ar structure (Leopo1d and Kriedemann, 1975).

Besides the mu1tip1e effects of cytokinins on nuc1eic acids andproteins synthesis, there are numerous instances where this hormonebrings about dramatic changes in the biosynthesis or the content ofother hormones; e.g., cytokinins may bring about increases in the auxincontent (Skoog an~ Armstrong, 1970), and gibbere11in content (Fuchs andLieberman, 1968).

vii. Gibbere11insThe gibbere11ins are a 1arge group of substances and at present

over forty different types have been characteri~ed. They are usua11yal1 diterpenoids but not a1ways possessing five rings and, according to

an agreed system of nomenc1ature, are termed GA1, GA2, GA3, etc. Froma structura1 point of view, GA9 may be regarded as the basic type anda11 others have the same framework but poses varying degrees of sub-stitution of side groups (See Figure 6). Gibbere11in GA3, termed thegibbere11ic acid, is one of the preve 1ant types in higher p1ants. Itisa1so be1ieved that some GA's are inactive "discard products" of activeforms (Leshem, 1973).

Leshem (1973) out1ined four possib1e modes of GA action: (a)nuc1eic acid metabo1ism - GA acting as a histone depressor or as anmRNA stabi1izer, (b) hormona1 action on ce11 membranes-increasing thepermeabi1ity of the membrane, (c) auxin inducer- hormona1 effect of-GAmay be indirect or mediated by 1M, and (d) "first messenger" in theadeny1 cyc1ase system.

The regu1atory roles of GA in p1ant growth appear to be stimu1a-tions of both ce11 division and ce11 en1argements; and in p1ant deve1op-ment inc1ude near1y a complete 1isting of deve1opmenta1 functions inp1ants from dormancy to reproduction and senescence (Leopo1d andKriedemann, 1975).

Several unidentified natural inhibitors have been suggested asGA antagonists, and the same is va1id for abscisic acid, ethy1ene andsome synthetic growth retardants. The possibi1ity of the regulation ofp1ant growth by natural or synthetic GA antagonists remains on1y aninteresting possibi1ity to date (Leopo1d and Kriedemann, 1975).

Street (1969) reviewed the 1iterature re1ating to the use of GAin tissue culture. The GA effects changed with the species, media" and

Figure 6: The structures of some p1ant gibbere11ins and suggestedinterconversions between them (Leopo1d and Kriedemann, 1975).

at-íl.

O

HO~

COOH Wl8· COàHoH GA1Z

+

Ho$M ~COOH. COOH

ahlHO~OH

COOH

HO~OH

COOHGA3

Gibberellic ocid

The design of the regulatory systems might reasonably includenot only means by which growth can be stimulated but also the means bywhich growth can be restrained. Hormonal systems which suppress growth

ethylene. A hormone system which appears to be much more widespread asa suppressor of growth is abscisic acid (ABA), an isoprenoid compoundwhich shares the mevalonic acid synthesis pathway with gibberellins andcytokinins (Leopold and Kriedemann, 1975).

Besides hormonal suppressions of growth, the plant has at hand awide range of secondary plant substances which accumulate in the plant.and have no apparent role in a metabolic sequence. In a wide range ofgrowth phenomena, these secondary plant chemicals serve as inhibitorsin the planto They may also be toxic to insects or browsing animals andthus serve to limit attacks on the plant or in other instances to act as

the plant that bears them, phenolic acids, phenolic lactones, and therelated flaviniums are the most common. A central theme in the area of

mineral deficiencies. The participation of inhibitors in the regu1ationof growth has been recognized since the late 1940's, and yet the physio-logica1understanding of inhibitors and their roles has deve10ped veryslow1y, especia11y in comparison with the understanding of auxins,gibberellins, and cytokinins (Leopo1d and Kriedemann, 1975).

ix. Abscisic AcidABA has the central position among the growth inhibitors. This

inhibitor can be readi1y obtained by a1coho1 extraction of many p1ant~teria1s. On a molar basis, ABA is active in rough1y the same rangeof concentrations as the auxins, gibbere11ins, cytokinins, and ethy1ene(Mi 1borrow, 1968).

The formation of abscisic acid may occur in part, through theoxidations of some xantophy11s such as vio1axanthoxin and xanthosin.Xanthosin, a keto derivative, may be an intermediate but a1so has bio1o-gica1activity. (See Figure 7) (Tay1or and Burden, 1970).

x. Pheno1ic InhibitorsThe pheno1ic acids from the best known group of inhibitors among

thesecondary p1ant chemica1s. Most of them are synthesized via theshikimicacid pathway, through the deamination of pheny1a1anine or~rosine, a1though a few are synthesized from acetic acid units.~mbers of the cinnamic acid series can be converted into the benzoicicidcounterparts by B-otidation (Zenk and Mueller, 1964). E1aborationlfmore comp1 icated hydroxy1 ation patterns can follow the formation of~e simp1e pheno1ic acids (Nair and Vining, 1965). The 1actonescoumarin

a~~·O~~ 'I

0=,1 COOH

MÁ\HOV Lo

series are formed by the ready laetonization of the phenylpropeneequivalents, espeeially after the formation of the o-glueoside (Neish,1965) and the eonversion of the trans-einnamie aeid to the eis eonfigura-tion by the aetion of light (Haskins and Gorz, 1961). The phenolielactones ordinarily are formed via hydrolysis of theglyeosides. Themore elaborated flaviniums and ehaleones (Figure 8) are thought to bederived from the einnamie aeid series with the addition of aeetate bloeks

plant tissues leads to the hydrolysis of phenolie glyeosides to theaglycones, whieh are markedly more reaetive (Pridham, 1965).

The inhibitory effeets of phenolies on growth are eommonlyattributed to the enhaneement of indoleaeetie oxidase, but it is veryprobable that other aetions sueh as an interferenee with the oxidativephosphorylation are involved (Pridham, 1965). The effeetiveness of

acting as stimulants of growth under some eonditions. The strueturalformulas of these inhibitors ean be seen in Figure 9.

Homologous flavinium derivatives sueh as kaempferol and quereetin(monophenolies and o-diphenolies respeetively) are likewise respeetivelystimulants and inhibitors of IAA oxidase. The polyhydroxy phenolies are

Figure 8: Some representative pheno1ic inhibitors of the f1avinium,cha1cone, and depside categories. Common location of gluco-side attachments are indicated by the G pointsrs (Leopo1d andKriedemann, 1975).

OH oM .@> HO{U-< >-OH

@> HO

mHO OH

H0-Y 6-'CV<~OH COOH

Chlorogenic c.cid

Figure 9: Some representative phenolic inhibitors. The origin fromphenyla1anine and tyrosine is indicated at the left (Leopo1dand Kriedemann, 1975).

Benzoic ocid series Cinnomic ocid series Loc tone series

O1NH2 (JCOOH (r 00~I COOH -( ~ I COOH ~ I o =0

Phenylolonine Benzoic ocid Cinnomic ocid Coumorin

mNH2 ~ -Q-COOH~

-O)HO ~ I COOH HO ~ I COOH HO ~ I o =0HO ~

Tyrosine p - Hydroxybenzoic ocid p - Coumoric ocid Umbelliferone

OH

O=COOH HO~ HO=óJHO ~ I COOH HO ~ I O =0~ OH

Solicylic ocid Coffeic ocid Aesculin

HO~COOH CH'O~ CH'o=OOHO ~ I COOHHO ~ HO ~ I =0O

OHGollic ocid Ferulic ocid Scopoletin

xi. Other Types of InhibitorsThe remaining inhibitors among the secondary plant chemicals are

divided among relatively small categories. Among the quinones, Juglone(Figure 10) is a well known representative. Related to the phenolicacids, quinic acid is of common occurrence, and is a component of thedepside chlorogenic acid. Intermediate between the lactones andquinones is chelidonic acid. The cyanogenic compounds, including themustard oils and mandelonitrile derivatives, occasionally occur in highconcentrations in some plants. Among the terpenes are many aromatic orvolatile compounds which serve as effective growth inhibitors (Bode,1939; Mueller et ~., 1964). Fatty acids include many natural growthinhibitors, which have received surprisingly little attention (Poidevic,1965). Amino acids may serve as growth inhibitors, e.g., hydroxypoline(Cleland, 1963) and naturally occurring analogs of amino acids such asmimosine (Suda, 1960; Smith and Fowden, 1966). Proteins or polypeptidesare occasionally reported as growth inhibitors (Elliott and Leopold,1953).

In several instances, plant extracts are reported to containsubstances which can inhibit the gibberellin stimulated growth (Koehlerand Lang, 1963; Michniewicz, 1967) or inhibit cytokinin stimulatedgrowth (Letham, 1963; Kefford et ~., 1968).

xii. Undefined Growth FactorsUndefined growth factors or natural produces are extracts of

plants or cells which are added to the media either with or withoutfurther treatment. This is usually in the hope of producing some

Figure 10: Some other types of growth inhibitors (Leopo1d andKriedemann, 1975).

unknown factor to facilitate the growth of material which is difficultto culture. Yeast extract is widely used in the culture of micro-organisms and is also used occasionally in plant tissue culture as asource of animo acids and vitamins. Casein hydrolysate is also used as anitrogen source occasionally. For rice, yeast extract was the mostsatisfactory nitrogen source followed by caS1n hydrolysate and thenammonium and nitrate media (Yatazawa and Furuhashi, 1968).

The most widely used and very effective plant extract is coconutmilk. To prepare this extract, either the liquid coconut milk("coconut water") is withdrawn and filtered and then used directly, orpart of the pul.p is ground up in a blender and added to the 1iquid. Someworkers autoclave the coconut mi1k in the medium while others fi1terthe coconut milk and add it separately to the medium (Sharp and Caldas,1972).

Original1y, coconut milk was used by Overbeek et ~., (1942) forthe culture of small Datura embryos. Since coconut milk is a naturalendosperm, it seemed a logical choice as a nutrient for the sma11difficult-to-culture embryos. Since then, it has found wide applicationin induction of callus and in morphogenesis of many species. Testingconcentrations from 0.5% to 30%, optimum fresh weight growth was found inpotato and sweet potato cal1us at 5% by volume coconut mi1k (Lingappa,1957). In carrot (Steward et ~., 1964) and Datura (Guha and Maheshwari,1967), coconut mi1k stimu1ated embryo formation, and it was the onlytreatment which successfully induced cell division in po11en grains ofBrassica of al1 hormone treatments tried (Kameya and Himata, 1970).

Coconut mi1k contains myo-inosito1, which stimu1ates growth ofca11us, often in a synergistic way with auxins. Coconut mi1k a1so hascytokinin activity, which can a1so interact with auxin to give enhancedgrowth. Thus, it is not surprising that cocunut mi1k interacts syner-gistica11y with auxins, for instance in the case of potato ca11us(Steward and Cap1in, 1951). The activity of coconut milk cannot becomp1ete1y attributed to these two factors, however, as cytokinin p1usinositol wi11 not comp1ete1y substitute for the effects of coconut mi1k.

Other p1ant extracts which have been used are carrot root forcarrot cal1us (Syono, 1965 and 1965 a), potato juice for potato ca11us(Lingappa, 1957), and toma to juice for tomato fruit cu1ture (Nitsch,1949). In the case of the potato cal1us, coconut milk was more effectivethan the potato juice and the tomato juice cou1d be rep1aced byphthoxyacetic acid. Various other extracts have been used from time totime such as waterme10n juice and prune juice (Guha and Maheshwari,1967).

xiii. Effectsof Growth Factors on Morphogenesi sThe morphogenesis of higher p1ants depends on the integration

and mutua1 interaction of various·organs, tissues and ce11s which areseparated from one another in space. Together they form a comp1ex systemfor ana1ysis of which it is necessary to know not on1y about thesubstances primari1y invo1ved in morphogenesis (for instànce, hormones),but a1so about the corre1ations which resu1t from the differentcapabi1ities, different p1ant tissues to synthesize specific compoundsand the transport of these substances. This system can be simp1ified

by isolating cells, tissues. and organs and their subsequent cultivationin ·vitro. This and the demonstrated persistence of the potentiality ofisolated cells and tissues (as manifested, for instance in the regenera-tion of leaves. roots,· flowers, and embryos) are the primary reasons forthe increasing use of cell and tissue cultures in morphogenetic studies.lhe use of such cultures implies the assumption that the processesinvolved in the formation of cells, meristems, organs and of the wholeplants are the same in vitro as they are in the intact planto

Morphogenesis of higher plants can be considered from a numberof quite different aspects. In the context of this review, it isappropriate that it should deal mainly with the initiation and develop-ment of organs and embryos in vitro and with the effects of growthfactors on these processes.

Gautheret (1945) was the first to show that auxin at optimalconcentrations could induce root primordia in carrot explants, but ina great number of subsequent investigations it turned out that aparticular inductive treatment developed for a particular culture doesnot guarantee its positive effect on other cultures. Auxin, for example,can inhibit root formation in carrot and pea culture and it can bewithout effect in tissues where it is not a limiting factor. Theentirety of these observations leaves no doubt that organogenesis invitro depends on a complex system of limiting and interacting factors.It is this situation which has led to different theories about the

regulation of organogenesis in vitro. One of these observations postu-lates that minimal quantitative changes in the ratio of certaincomponents of the nutrient media are decisive and determine whetherorgans are initiated ar noto The classical example for such a regulationis seen in tissues derived from the pith parenchyma of tabacco shoots(Skoog and Miller, 1957). When this tussue is isolated and grown invitro, cell division and true growth occur in a synthetic media only ifit contains auxin and kinetin ar some other effective cytokinin. Theconcentrations of these two substances determine both the qualitativeand quantitative aspects of tissue growth. On an agar medium containing2 mg/l of auxin and 0.1 mg/l kinetin, only callus proliferation occurs.The two substances in this case work additively. However, if thekinetin concentration is lowered to 0.02 mg/l without altering theauxin level, root morphogenesis is induced. Higher concentrations ofkinetin (0.5 mg/l lead, conversely, to the initiation of shoots; rootformation is then supressed. Other components interfere in this inter-action. Increasing the phosphate concentration of the culture medium canreinforce the shoot formation and suppress or weaken the root promotingeffect of auxin. Adenine and amino acids, such as tyrosine, actsimilarly, for in suitable concentrations they augment the kinetin effectand reduce that of auxin (Reinert, 1973).

So far, there are only a few observations with tissues other thantobacco supporting the principle that organogenesis is regulated byquantétive shifts in the ratio of hormones and other substances. One ofthese pertains to chicory which the zone of shoot formation contains arelatively large amount of native cytokinin, whereas auxin predominates

in the root forming region (Vardjan and Nitsch, 1961). Experimentswith tissues from cyclamen tubers have also shown interactions betweenauxin and purine derivatives (adenine, guanine) in the regulation ofroot and shoot formation. But the decisive factor for root or shootformation in these cultures apparently was not the ratio between thesesubstances, but rather the absolute concentration of the auxin in themedium (Stichel, 1959). In cultures of a variety of Amoracea. rusticanait was even possible to induce shoot formation by raising the auxinconcentration and to inhibit it by adding kinetin (Sastri, 1963).Kinetin at physiological concentrations also blocks morphogenesis incarrot cultures (Reinert, 1959). Thus it could be that cultures fromthe medullary parenchyma of tobacco shoots are a somewhat special caseso that the principle of regulation of organogenesis by quantitativechange of the ratio between certain specific compounds cannot begeneralized to cover cultured tissue in general (Reinert, 1973).

It is equally impossible to deduce from the limited number ofexperimental data currently available on flower formation in tissueculture as to whether this process is controlled by quantitative orqualitative changes. The factors that have been found to be essentialfor the initiation of flower primordia in explants oftobacco (Aghion-Prat, 1965), Plumbago indica (Nitsch, 1968) and Lunaria annua (Pierick,1967) are apart from the light involved in photoperiodic induction andthe low temperature in vernalization or relatively high level ofnitrogen in the medium, plus cytokinin and various constituents ofnucleic acid (adenine and orotic acid). Auxins, gibberellins and variousorganic nitrogen compounds (with the exception of urea) had an inhibitory

effect. A quantative relationship between these factors, similar tothose between auxin and kinetin in root and 'shoot formation in tobacco

formation. In this work, the inhibiting action of lAA on flower forma-tion in tobacco callus could be counteracted by adding different nucleicacid base analogues (Skoog, 1971). Although these results are part1ycontradictory, they do not exclude the possibi1ity that the reaction

.differing on1y in that different limiting factors are decisive fordifferent species or fami1ies (Reinert, 1973).

It is not clear whether inorganic nitrogen compounds or hormoneslike auxin and cytokinin induce organogenesis in cell cu1tures de novo,or whether they enhance merely a process which begins already with theisolation of cells. Almost nothing is known about the mechanism ofaction of these substances. If this background is taken into accountit is not surprising that recently the rhizocaline theory has beenrevived. This theory imp1ies that not quantitative but rather qualita-tive changes, i.e., the synthesis of the hypothetica1 rhizocalinedetermines the initiation and the development of roots. The basis ofthis concept is experimental data on the root formation of cu1tures ofHelianthus tuberosus indicating that in addition to auxin and sugar,temperature and 1ight are essentia1 factors and p1ay a decisive role inthe formation of the rhizocaline in cultures (Gautheret, 1966).

synthesis of other organ specific substances in shoot and leaf produc-tion, i.e., substances like caulocaline and phyllocaline.

The first clear cut proof of the induction and development ofembryos in carrot tissue was achieved by a succession of changes innutrient media. The decisive step in these experiments was the transferfrom auxin-containing to auxin-free media and the addition of nitrogenin the form of amino acids (Reinert, 1959). These observations weresubsequently confirmed and it was discovered that embryogeneis in vitrocan be induced by both inorganic (KN03, NH4N03) and organic nitrogencompounds such as amino acids and amides (Tazawa and Reinert, 1969). Itis now also clear that in order to initiate embryogenesis, most saltsmust be present in high concentrations (Butenko et ~., 1967) whereascompounds like ammonium (Halperin, 1966) and glutamine are effective atvery low leve1s. However, these substances are not specific for embryo-genesis l~ vitro; in other words, they are interchangeable. Thus thenon-inductive medium prepared according to White's formula (1954) whichcontains on1y 3.2 nM nitrogen, can be converted into an inductive formby the addition of only 5 mM glutamine. If, instead of the amide, KN03is added to the same medium, a re1atively high concentration (40 mM) isrequired to produce the same effect. It is not the absolute quantity ofthe nitrogen which determines the triggering of embryogenesis in vitro,although the ratio of nitrogen to auxin is important; embryo formationcan be triggered on White's medium in spite of the low nitrogen content(3.2 mM) by omitting auxin (Reinert, 1973).

The significance of the successive changes in the nitrient mediamentioned above for the process of embryo formation in vitro has beenconfirmed with tissues other than carrot (Steward et~., 1966), and notonly for embryogenesis but a1so for organogenesis (Torrey, 19~6) andcytodifferentiation (Fosket and Torrey, 1969).

Steward et ~., (1966) assumed that coconut mi1k (CCM) isessentia1 for the initiation and deve10pment of carrot embryos in vitro.Later on, it was shown by several workers that ce11s from carrotcu1tures are ab1e to form embryos on synthetic, chemica11y defined mediawithout CCM; indeed, CCM and also cytokinins can even inhibit embryogen-sis in vitro. CCM and cytokinins obvious1y stimu1ate on1y after lateembryo stages have been formed (Reinert, 1963; Ha1perin and Wetherall,1964) .

2. Tree Tissue Cu1tureAbout 220 woody species have been used for tissue cu1ture techni-

ques, but so far, on1y a few species have been reproduced by thosetechniques. There are no general rules of nutrition or deve10pmentalcontro1 that can be app1ied even to c10se related species, and most workis empirica1 in nature. The motto seems to be Jlif it works, use it.1I

However, the long term goa1 must be the understanding of the biochemicaldifferences between undifferentiated and differentiated cel1s among manytree species, and then perhaps one can start to formulàte some generalre1ationships that must be met before organogenesis can be triggered.

The trigger at the molecu1ar level may be identica1 for manyclasses of organisms, but one may discover that the same criteria may be

metal ions, nitrogen forms and levels, and perhaps as yet unknown effectsproduced by combining all of these with sugars, vitamins, and othersupplements. The time has come to shift the major attention to morebiochemical studies, as it has been done with many tree tissue culturesalready, but one must also be able to translate this basic informationinto meaningful feedback for concomitant empirical experiments (Winton

The use of tissue culture techniques is rapidly expanding as anew approach to solving biological prob1ems in woody species, particu;1arly trees. Somatic and haploid tissue culture, protoplast culture,embryo cu1ture, meristem cu1ture and ~dal culture have been used to

1\

a. GymnospermsThe pioneering efforts to initiate ca11us from isolated tree

exp1ants were reported by Gautheret (1934). Ca11us grew on cambi a1exp1ants col1ected from the trunk of Abies a1ba and Pinus pinaster, butthe cal1us grew slowly and died after a few months.

The first continuous cu1ture of conifer cal1us was obtained byBall (1950) from Seguoia sempervirens. Buds grew from ca11us during the..:.-

""",,,v3-5 subculture, and then only from c1ear ce1ls that did~contain tannin.P1antlets have been reported from ca11us of Ginkgo bi10ba by Hackett(1964) and from Ephedra fo1iata by Sankh1a and Sankh1a (1967). Rootswere grown frem Ginkgo bi10ba haploid tissue by Tulecke (1967) andTukecke et ~., (1962).

Tab1e 2: Tree species used in tissue cu1ture regenerating roots, shoots,p1ant1ets and asexua1 embryos.

Gymnosperms:Ephedra fo1iata

Seguoia sempervirensPicae abies

Pinus pa1ustrisPinus rigidaPinus strobus

Straus (1962) and Sankha1a P1ant1etsand Sankha1a (1967)Sussex and C1utter (1959)and Tu1ecke et ~., (1967) RootsTu1ecke (1967) andHackett (unpub1ished)Ba11 (1950) ShootsHuhtinen (unpub1ished) andChalupa (unpublished) ShootsDurzan et a1., (1973) andChafe and Durzan (1973) Asexua1 embryosKonar and Oberoi (1965) Asexua1 embryosIshikawa (1967, 1972,1973 and In Press) Roots and ShootsBer1yn (unpub1ished) Roots and ShootsBer1yn (unpub1ished) Roots and ShootsWinton (unpub1ished) Roots and ShootsWinton (unpublished) Asexua1 embryosSommer et ~., (1975)Sommer et ~., (1975)Sommer et ~., (1975)Sommer et ~., (1975)Isikawa et ~., (1967)Ber1yn (unpub1ished)

Asexua1 embryosAsexua1 embryos

79

Species Workers Regeneration

Pinus taeda Sommer et a1., (1975) Asexua1 embryosand Winton-Yunpub1ished)

Pseudotsuga menziesii Winton (unpub1ished) Asexua1 embryosTsuga heterophy11a . Winton (unpub 1ished) Asexua1 embryosAngiospermsBetu1a pendu1a Jacquiot (1955, 1959 a

and b, and 1964) RootsBroussoneta kasinioki Oka and Ohyama (1972) P1ant1ets

Ohyma (1972) RootsCitrus auxantium Esan (1973) Asexua1 embryosCitrus grandis Esan (1973) Asexua1 embryosCitrus hystrix Esan (1973) Asexua1 embryosCitrus ichangensi s Esan (1973) Asexua1 embryosCitrus jambhiri Esan (1973) Asexua1 embryosCitrus kharna Esan (1973) Asexua1 embryosCitrus 1ansium Esan (1973) Asexua1 embryosCitrus 1emon Esan (1973) Asexua1 embryosCitrus 1imoni a Esan (1973) Asexua1 embryosCitrus madurensis Esan (1973) Asexua1 embryosCitrus medica Esan (1973) Asexua1 embryosCitrus paradi si Esan (1973) Asexua1 embryosCitrus reticu1ata Esan (1973) Asexua1 embryosCitrus sinensis Esan (1973), Mitra and

Chaturverdi (1972) andKochba and Spiege1-Roy(1972 ) Asexua1 embryos

Coffea canephora Staritsky (1970) Asexua1 embryosDiospyros kaki Yokoyama (1973) Roots

Euca1yptus citriodora Aneja and Ata1 (1969)Euca1yptus grandis de Fossard (1974)G1edistsia triacanthos Rogozinska (1967 and

1968)Hevea brasi1iensis Paranjothy (unpub1ished)I1ex aguifo1ium Hu and Sussex (1971)Ma1us sy1vestris Okuse (1968)Morus a1ba Ohyama (1970) and Ghuga1e

et -ª.l., (1971)Oshigane (1973)Lavee (unpub1ished)Schroeder (1955, 1957,1959, 1961, 1962, and1963)Mathes (1964 c)Mathes (1964 c) andTari s (1966)

Popu1us a1baPopu1us a1ba .!CanandensisPopu1us canadensis.! grandidentataPopu1us canandensisx tremu10ides Winton (unpub1ished)

Berbee et -ª.l., (l972)Ghuga1e et ~., (l97l)Popu1us nigra

Popu1us tremu10idesx alba

Winton and Einspahr (1968)and Winton (unpub1ished) P1ant1ets

pa1ustris, Pinus e11iottii, Pinus taeda and Pinus rigida, which gaverise to vigorous p1ants.

Winton (1972 a and b, 1973 and 1974) reviewed 1iterature cita-tions and discussed 51 gymnosperm species which have been used in tissueculture.

Embryos were a1so found in cell suspensi ons of Pinus strobus byIsikawa et ~., (1963) and a1so in suspension cu1tures of Picea glaucaby Dunzan et~., (1973) and Chafe and Dunzan (1973).

b. AngiospermsTrip10id Popu1us tremu10ides, tetraploid f.. tremula, and dip10id

hybrid between f.. tremu10ids and f.. a1ba have been reproduced fromundifferentiated somatic ca11us cu1tures (Winton, 1972 and 1973). Shootswere produced by Wo1ter (1968) and Mathes (1964) from Popu1us tremu10idesca11us. These shoots were excised and rooted to form p1ants.

P1ant1ets were produced from 1ignotuber tissue of Euca1yptuscitriodora by Aneja and Ata1 (1969).

Jacquiot (1966) was successfu1 in producing p1ant1ets from U1musglabra ca11us.

Hu and Sussex (1971) produced embryos from coty1edons of I1ex

~Roots were produced in tissue cu1ture of Betu1a pendu1a (Jacquiot,1955, 1959 a and b, and 1964); Broussoneta kazinioki (Ohyama, 1972);Diospyros kaki (Yokoyama, 1973); Euca1yptus grandis (de Fossard, 1974);Ma1us sy1vestris (Okuse, 1968); Morus a1ba (Ohyama, 1970) and Ghuga1e

et -ª.l., (1971); Persea americana (Schroeder, 1955, 1957, 1959, 1961, 1962and 1963); Popu1us nigra (Ghuga1e et -ª.l., 1971) ano of Ti1ia cordata(Jacquiot, 1951) and (Bychenkova, 1964).

Shoots were produced to a 1esser extent than roots in threetissue cu1tures: G1edistsia triancanthos (Rogozinska, 1967 and 1968);Morus a1ba (Oshigane, 1973) and Popu1us euroamerica (Berbee et -ª.l.,

Trees were mass produced from stem-tip ca11us by Berber et -ª.l.,(1972), from Popu1us canadensis when they were trying to obtain virus-free material.

Citrus trees were produced from nuce11us and ovu1e by Esan (1973).These nucelar iso1ates were deve10ped into adventive embryos in cultureand after that were subcu1tured to form p1ants that were p1anted in soi1.

Orthotrophic shoots of Coffea arabica, C. canephora and f. 1iber-ica were cultured by Staritsky (1970). Embryo and p1ant1et formationwere observed only in f. canephora tissue, whereas on1y fast growingca11us ce11s were obtained in the other two species. The globu1arembryos formed in f. canephora tissueswere de1imited to the periphery ofthe ca11us. The deve10pment of p1ant1ets in one species out of threec1early suggests the existence of different potentialities among differentcoffee species (Monaco et -ª.l., In Press).

Sato (1974), culturing anthers of Populus deltoides, ~' maximo-wiczii and f. sieboldii ~ grandidentata obtained shoots and .plants, andthe diploid chromosome number was observed in root tips of the normalplantlets. Haploids were not recognized.

3. Eucalyptus Tissue CultureThe cultivation of Eucalyptus has presented many problems to

researchers in their efforts to use known methods and techniques ofpropogation. Past work included the use of different parts of organssuch as roots, lignotubers, stems, and hypocotyls; all from a widevariety of different Eucalyptus species. Success was noted in theregeneration of plantlets from lignotubers of Eucalyptus citriodora androoting of stem segments of Eucalyptus grandis. Major uses ofEucalyptus tissue culture techniques have been directed towards theachievement of vegetative propogation with some selected uses for cytolo-gical studies. Attempts at successful regeneration included experimentswith different culture media containing defined and undefined growthfactors, mineral salts, andphenols. Until the present, no work has beennoted with Eucalyptus leaves or anthers as a source of material fortissue culture techniques.

Bachelard and Stowe (1963) studied the growth in vitro of rootsof Eucalyptus camaldulensis testing the effects of seed extracts, aminoacids, "chaff," yeast extract, shikimic acid, mevalonic acid, NAA andcoconut milk. The latter was considered to be essential for the growthof roots, an unusual requirement for an organic culture. A piece ofhypocotyl tissue had some beneficial effect on growth but it did notcause the roots to grow adequately. Yeast extract inhibited the growthof completely isolated roots in culture medium containing coconut milk.

Extensive studies were conducted on cambial activity, tissueorganization, and organogenesis, in which cambial tissue from twentycommon forest tree species was cultured. Although it was always possible

to obtain growth of explants, continuous cu1ture on synthetic mediasucceeded only with aspen, birch, chestnut, lime, elm, and wild cherry.Five Eucalyptus species (I. grunni, I. tereticornis, f. caldocalix,I. gamocephala and f. camaldulensis) required coconut milk in the mediaand grew very slowly (Jacquiot, 1964).

Marcavillaca and Montaldi (1964) used explants of I. trabutistem wood where callus was produced mainly by proliferation of the woodray parenchyma, and also from cambial and interphlaem cells. Stem woodpieces produced isolated roots only in some f1asks with media containinghigher concentrations of lAA after several weeks of culture. Theresearchers failed to obtain shoots in experiments with kinetin and IAA.Aqueous and alcoholic extracts of different organs of the tree, coconutmilk, yeast extract, and casein hydro1ysate used in experiments fai1edto induce organogenesis or to improve the ca11us growth.

Cronshaw (1965) used Eucalyptus camaldu1ensis cells frbm ce1lsuspension cu1ture to study ce11 wa11 development and cytop1asmaticfine structure. Sing1e membrane bounded crystal containing bodies werefound in association with tannin.

Sussex (1965) used hypocoty1 segments or strips of superficialtissue of hypocoty1s consisting of epiderma1 and cortica1 parenchymace11s frem Euca1yptus cama1dulensis seedlings in White's medium (White,1943) supp1emented with 2,4-0 and autoc1aved coconut milk. He obtainedfriab1e ca11us which gave origin to free cel1s and multicel1ular aggre-gates that were screened and subcu1tured up to 36 passages over a threeyear period. The diploidy level (2n = 22) was kept constant during thisperiod. If 2,4-0 was omitted from the medi um, much of the tissue died.

The growth of different separated ce11s or aggregate of ce11s wasstudied and measured week1y.

Aneja and Ata1 (1969)s using a mediumnot very we11 describedand exp1ants from stem tissue and 1ignotuber tissue of ~. citriodoraswere successfu1 in obtaining bud-1ike pe11ets from stem tissue ca11usand p1ant1ets from 1ignotuber tissue ca11us.

Fine (1960) describes the tracheary e1ements formed inE. rostratas and discusses the effects of IAAs 2s4-Ds ca1cium or EDTA one1ement differentiation and the resu1ts of attempts to measure the1ignotuber content and amounts of peroxidase present.

Piton (1969) studied the process of ce11 divislon and theformu1ation of ce11 membranes in tissue cu1ture of E. cama1du1ensia.

B1ake (1972) found root cal1us deve10ped from Euca1yptus1ignotubers into a teratomaceousmass of roots and shoots that' cou1d besubcu1tured to produce who1e p1ants of ~. ob1iqua and E. vimina1is.Howevers Winton (1972 b) fai1ed to produce organs from stems capsu1e-wa11 or 1ignotuber tissue from Euca1yptus nova ang1ica.

De Fossard et ~., (1972) reported that tissue cu1ture of fivespecies of Euca1yptus were estab1isheds a11 on coconut free media andthe success was 1imited to some root regeneration.

Kitahara and Caldas (unpub1ished) studied the deve10pment andgrowth of ca11us from hypocoty1 exp1ants of Euca1yptus a1bas ~. grandis,and~. citriodora using White's 1963 medium with coconut mi1k and/or2,4-D and lAA in different concentrations obtaining satisfactory resu1tson ca11us growth.

E. grandis. Nodal explants from seedlings were used in the experiments.Rooting was most readily achieved with explants from the base of seed-lings; best results were obtained with foliated nodal explants.

Or. Fossard (1974), using a medium devoid of coconut milk,ctlltured seedling stem explants of ~. bancroftii, I. laevopinea,I. melliodoka and I. grandis. One of these species, E. bancroftii, grewvigorously on a completely defined medium; the other four required caseinhydrolysate for rapid growth. Roots were induced to regenerate fromundifferentiated callus of E. grandis on media containing either 2,4-0or various combinations ofIAA and kinetin.

Lee and de Fossard (1974) studied the effects of various auxins(IAA, NAA, 2,4-0, NOA) and cytokinnins (kinetin and BAP) on in vitroculture of stem and lignotuber tissues of ~. bancroftii in an attempt toinduce regeneration of organs. Regeneration was not achieved, butsubstantial differences in growth were obtained. Best growth occurred onbasal medium with 2 x lO-5M NAA and 16 x 16-6M BAP (for stem calluses)and 2 x lO-5M 2,4-0 and 2 x lO-6M BAP (for lignotuber calluses).

De Fossard et·~., (1974) summarized the responses of callusesto variousauxin and cytokinin modifications of the culture medium fromtissue culture of stems from young and adult trees of E. grandi s,~. nicholi, ~. laevopinea, I. bancroftii, f. milliodora,·and I. urnigera,and lignotubers of I. bancroftii. Regeneration of pla'nts from suchcalluses was not achieved. A cultural mixture of Eucalyptus callus andregenerating tobacco callus did not induce regeneration of Eucalyptus.Organ culture of ~. grandis is described. Axilary bud development and

seedlings, particularly with foliated explants. Successful root develoo-pent of plants from nodal culture was also achieved with older(upto 7months) ~. grandis trees, using a cu1ture medium with 5 x 10-6Mindolbutyric acid. These p1ants had more than 50 nades, and organcu1tures were initiated from nades considerably higher then the 15thnade IIbarrierllexperienced with c1assica1 methods of propogation (Paton

c. Juveni1ity Factors in Woody P1antsIt has been known that in many plants there are considerable

differences between the early ar juvenile and the adult stages ofdeve1opment. This distinction between juvenile and adult phases isfound in al1 groups of p1ants, but is perhaps more familiar in the

the shape of the successive 1eaves of the individual seed1ing arspore1ing and other morpho1ogical and physiological changes. Much ofthe know1édge on this subject has been supp1ied by the extensive investi-gations of Goebe1 and his co11eagues, which have been summarized insuccessive editions of the lIorganographic der Pflanzenll Goebe1 (1898 and1928). More recent1y, Ashby (1948) has confirmed Goebel's work onheterob1astic deve10pment on a more ana1ytica1 basis.

The views of Goebe1 have not gane unchallanged, but recentexperimental studies have indicated that his treatment of the prob1em isbasical1y sound and requires only comparatively slight modification andextension to retain its va1ue in the interpretation of wide range of

contemporary reassessment of Goebel's hypothesis may be desirable,particularly since his conclusions are often ignored or rejected incurrent botanical literature. Goebel's principal contribution was arealization of the importance of nutrition in regard to aging.

Many morphologists of his period considered that juvenile leavesref1ect a re1atively primitive state, so that the ontogeny of theindividual p1ant is to some extent a recapitu1ation of phy1ogeny. Goebelmaintained, however, that although in some cases, for examp1e, certainspecies of Acacia, the juveni1e 1eaves may correspond to the adultleaves of a phylogenetica11y ear1ier species, in a majority of p1antsthe change in form of successive leaves is no more than a response tothe increasing nutritional powers of the germ1ing. He he1d that a1l1eaves of the plant conform to the same pattern of development, but thatin juvenile 1eaves the deve10pment is arrested at one or other of theintermediate stages of the complete sequence necessary for the attain-ment of the adu1t forms. The later-formed 1eaves are checked at pro-gressive1y late stages of deve1opment; consequently, when arranged inorder they repeat fairly close1y the deve1opmenta1 sequence characteris-tic of the individual adult leaf. This principle was rediscovered byBrown (1944) in his study of leaf development in larkspur.

If juvenile 1eaves are arrested organs, it might be expectedthat there would be a reversion to such leaves in adu1t p1ants exposedto starvation conditions, and numerous examp1es of thf~ kind have 1ndeedbeen described by Goebel (1898 and 1928).

Many other workers have contributed to the great body ofevidence in support of a connection among heterob1astic deve1opment,

10ss of apical dominance and other factors correlated with juvenilityand nutrition, but much of this evidence, although convincing in itscombined impact, has suffered from the disadvantage that the methodsemployed for changing the nutritional status of the experimental plantshave usually involved changes in other factors, such as lighting,temperature and humidity, which may be critical from the present pointof view.

i. Some Morphological and Physiological ChangesRobbins (1957) reported that Hedera helix from seed grows as a

vine with five lobed leaves in a 2/2 ranking. It is plagiotropic andcrawls along the ground, rooting at intervals. If it reaches a wall ortree trunk it grows up the support, clinging to it with aerial roots.Sooner or later the upper portion of the vine changes in growth habitoOval leaves with entire margins are produced in 2/5, 2/6 and 2/7 ranking;the growth is orthotropic, aerial roots are no larger produced, and thisportion of the plant blooms and fruits.

It has been reported that in Acacia melanoxylon seedling plantthe bipinnate leaves at the base are characteristic of the juvenilecondition; the uleaves,1I which are actually expanded petioles (phyllodia),at the top of the seedling are characteristic of the adult form, andthere is a gradual change in the leaves from base to topo This issimilar in Eucalyptus, in which juvenile leaves at the base are large,broad, and have no petiole (sessile); mature leaves on the upper part ofthe shoots are elongated and have a distinct petiole (Hartman and Kester,1968).

Moorby and Wareing (1963) studied the changes occurring in Pinussylvestris and Larix leptolepis during ageing and reported an investiga-tion into the possible mechanisms controlling these changes, and con-cluded that such a reduced annual growth increment, loss of apicaldominance and changes in the geotropic responses are easily reversibleby pruning or by grafting shoots from older trees on to young stocks, andthese changes are referred as 'ageing'. Pruning experiments suggestedthat ageing is due to increased competition for nutrients between thevarious constituent shoots as the branch systems increase in complexity,but the distribution of available nutrients is modified by spicaldominance.

It has been cited in the literature the growth and deve10pmentof straight unforked stems of tropical species during the juvenile phaseof growth of trees, e.g., Gram and Larsen (1960).

The wel1 defined juveni1e character in Fagus si1vatica wasphases in the reproductive 1ife of the tree. As was pointed out byMue11er (1937) and Wareing (1958), when the Pinus nigra and Pinussy1vestris tree have reached an age of 5-7 years it starts to formfemale cones, and these appear on the vigorous distal parts of thebranches and on the "1eader.1I .The male cones are not formed unti1 thetree is from 10 - 15 years old, and they are then formed on the weakertwigs in the proxima1 parts of the branches.

Wareing (1959) pointed out an apparent rough correlation betweenthe juveni1e period of a given species and its normal maximum size atmaturity. Once a given tree starts f10wering, it normally continues tof10wer every year (Crossley, 1956), but in some species, such as Fagus

silvatica, flowering appears to be very sensitive to weather and mayoccur at irregular intervals.

Wareing and Robinson (1963) developed experiments demonstratingthe importance of the "size factor" in the attainment of the floweringcondition by seedlings trees. Flowering was successfully obtained withseedlings Japanese larch in their 5th year by first growing the plantsrapidlyin a greenhouse until they attained a height of 9 - 10 ft. andthen planting them in the open in a horizontal position to promoteflower initiation.

Robinson & Wareing (1968) reported that flowering first occursafter the phase change has taken place. Flowering of Betula verrucosaoccurred much sooner in seedlings grown continuously under long daysin a g.reenhouse (i.e., wi thout donnant peri ods) than in those grownunder alternating periods·of longand short days, with chilling in thegreenhouse, or in pots in the open. Early flowering of larix leptolepisand Ribes nigrum was obtained when appropriate flower inducing treatmentswere applied. Experiments with R. nigrum showed that size of plantalone is not a primary factor in detennining the occurrence of the phasechange.

A lot of experimentstrying to propagate vegetatively the adultcondition have failed and it is explained as being the reversion of theadult condition to the juvenile, or the difficulty when rooting thecuttings.

Woycicky's (1954) experiments to find whether juvenile forms ofThuja occidentalis and T. orientalis can be perpetuated by means ofcuttings from seedling shoots with needle leaves, gave negative results,

because cuttings passed through the normal stages of foilage development.It seems likely therefore that the Retinospora forms of this species andof Chamaecyparis species have risen spontaneously as 'sports' eitheras seedlings that retain their juvenile form or shoots with juvenilefoilage that appear from time to time on mature specimens.

Schaffalitzki de Muchadell (1956) reported that leaf-sheedingtips from 5 to 6 years old beech clearly showing the juvenile character-istic of retaining withered leavest were used as scions with basal partsof woody scions grafted on to seedlings root-stockst and shoot tipsgrafted on to old stock. In both cases grafts had retained their leaves.

Wareing and Robinson (1963) reports on a series of graftingexperiments suggesting that scions taken from juvenile trees and graftedon to mature trees flower more readily when derived from longer seedling(which are approaching the mature condition) than from smaller ones.

Moorby and Wareing (1963) studied the correlation betweendegree of ageing and coning in grafts. The behavior of the graftsillustrates the fact that although the potentiality for coning is amaturation changet i.e.t once it has been attained it is not lostt itsobvious expression in the formation of male cones is under the controlof aging changes. This can be seen by comparing the behaviort aftergraftingt of twigs which had previously borne male or female cones. Ifa twig which is bearing female cones is used as scion and grafted on toa seedling stock it continues to produce female cones for a few yearstand gradually increases in vigor to give a strongly growing planto Nomale conest howevert are produced for several years until the graft hasproduced aged shoots and it is on these that the male cones are eventually

produced. Similarly, when a twig bearing male cone is used as scion itrapidly increases in vigor. It soon ceases to produce male conesand produces instead, female cones, and its further devélopment is fromthat point, similar to that of the f~male scion.

Muzik and Cruzado (1958) studied the transmission of juvenilerooting ability from seedlings to adults of Hevea brasiliensis bygrafting buds from a mature clone on seedling plants.

In experiments to propagate juvenile forms by taking cuttingsfrom juvenile branches of Chamaecyparis pisifera, scale-like leaveseventually developed on all the cuttings, except when there happened tobe a mutant that would have retained the juvenile form when left onthe parent tree (Langner, 1964).

Many studies were reported on rooting promotors and inhibitorsin cuttings of apple and plum (Challenger et ~., (1964); Girouard1066,1967, and 1968), Hess(1957, 1959, 1961, 1962 a, b, c, 1964,1965 and 1966) in Hidera helix; Paton et ~., (1970) in Eucalyptusdeglupta and ~. grandis). In a general way the cuttings from juvenilefoliage are easy-rooting and have promotors in some cases identified asphenolics compounds (alkaloids - as chlorogenic isochlorogenic, andquinic acids); rooting inhibitors are correlated with tannins, and theyare present in cuttings from the adult foliage.

Kranz (1931) suggested that the horticultural variety of Hederamight sometimes be vegetatively propagated in intermediate forms whichretained their characters just as propagations from the adult stage do.He suggested that the variety Iglymi" was a topophytic form, interme-diate between juvenile and adult. He noted that some cuttings taken

from the transitiona1 zone between the juveni1e and adu1t stage deve10ped1ike the variety IIG1ymill

; the new growth on others taken from near wherethe juveni1e form dominated and reverted to the juveni1e form whi1ethe new growth of cuttings near where adu1t fo1iage dominated was adu1tin character.

Robbins (1959) observed in Hedera he1ix that pruning causedsome p1ants to deve10p branches with mixtures of juveni1e and adu1tcharacters; this exp1ains why other workers got juveni1e shoots bypruning. He conc1uded that juveni1e shoots induced by pruning may bethe deve10pment of dormant buds from the 10wer portion of the trunk of aseed1ing tree which is considered to have retained its juveni1e charac-ter even though the upper portion of the p1ant is adulto They may a1sodeve10p from adventitions buds induced on the adu1t portion of theplanto

ii. Factors Which Induce Reversion of the Adu1t to a Juveni1e ConditionA number of studies have been undertaken to exp1ain the rever-

sion of the adu1t to a juveni1e condition and vice-versa, but even1imited successeshave not been obtained in the second case.

Hormona1 or environmenta1 factors have been able to inducereversion of the adu1t to the juveni1e condition.

Robbins (1957 a, b, 1958) conc1uded that gibbere11ic acid hadinduced a reversion of the adu1t ivy to a juveni1e condition.

Bochert (1965) reported that gibbera11ic acid treatment app1iedto the apica1 meristens in Acacia me1anoxy1on caused reversion frommature to juveni1e 1eaves.

Scurfield and Moore (1958) reported that application of gib-berellic acid prornoted the process of ageing in Eucalyptus rnelliodora.The effect of gibberellic acid was different frorn that obtained byother workers.

Trippi (1963) reported that GA sprays prevented leaf abscission,and caused reversion to the juvenile leaf-forrn on 3 year Acacia melano-xylon.

Corcoran et ~., (1972) studied the antagonistic effect oftannins and gibberellic acid in cucurnber seedlings and obtained differ-ent results for the different hydrosoluble tannins tested.

Kranz (1931) reported that cld plants of Hedera helix which hadbeen severely frozen back in a hard winter in Gerrnany formed juvenileshoots frorn the adult portion of the adult portion of the plant whengrowth was renewed in spring. He believed that these juvenile shootscarne frorndormant buds which were juvenile because they were originallyproduced when the plant was juvenile.

Robbins (1959) reported that the apical rneristernsare the sitesin which stages frorn juvenility to the adult state originate.

Juvenility appears to be an unstable metabolic state whichproceeds through a series of steps to a relatively steady state of theadult rneristern.·Later the adult rneristernrnay be reversed towardsjuvenility by treatment with cold,x-rays, gibberellic acid, severepruning, products frorn the juvenile state, formation of adventitiousrneristerns,forrnation of nucellar ernbryos, and by sexual reproduction.It is suggested that the two kinds of rneristerndiffer in possessingparticular rnetabolites that determine the rnetabolic systerns responsible

for the differenees in physiology and morphology.Brian (1953) reported the role of gibberellin-like hormones in

regulation of plant growth and flowering and that red light ean promoteproduetion of gibberellin~like hormone.

Under this topic we can consider the nutritional conditions

understand more ofthem to learn something about the phenolics com-pounds and their metabolism and role.

Changes in nucleie acids assoeiated with maturation andsenescenee in Hedera helix were reported by Millikan and GhoshThey concluded that signifieant changes in nueleic acids are associatedwith both maturation and senescenee of Hedera helix leaf tissues.DNA eontent was unaffected by senescenee, and there were no quantitativedifferenees between juvenile and transitional forms. The adult tissue,

was reduced by senescence in juvenile and transitional tissue but not inadulto Maturation, on the other hand, progressively deereased the

tive differenees in the nueleotide analysis suggest that different sRNAand rRNA species are responsible for the biological processes assoei ateswith maturation.

iii. Use of Tissue Culture to learn More AboutControl Mechanisms and Juvenility

The possibilities of the tissue eulture use to learn about control

Stoutemyer and Britt (1969) established tissue culture fromboth juvenile and adult stems of Hedera helix in White's mediumsupplemented by coconut water and auxin (NAA). With repeated transfers,cultures were habituated in less than a year to grow well without

seedlings were less demanding in requirements for growth. In a11 typesof cultures occasionally small areas of rapid1y growing cel1s werenoticed. These when isolated, gave rise to rapidly growing cultureswith many cel1s of unusual appearance. Abnormally 10ng cel1s and

I

chain-type ce11s were abundant. When 0.1 mg/1 of kinetinr was added tothe medium, these ce11s grew we11 without auxin and coconut water.

Robbins and Harvey (1970) reported that cal1us from stem piecesof the juveni1e and adult stages of Hedera he1ix were grown for 17

coconut water and for a fewer passages on media which lacked coconutwater. Seedling callus differed in its growth in culture from adulto

callus to adapt to unfavorable media was greater. On low salt mediaroot formation by callus was infrequent. Adult callusses formed rootsmore freely on high salt media than seedling calluses did.

The explants used in the experiments were from organs or partsof organs of commercially important Eucalyptus species in Brazilianforests. Explants used included: seeds, segments of seedling roots,hypocotyls, cotyledons, modal and non-modal stem segments, petioles,leafblade segments, apical shoots and anthers. The Eucalyptus speciesused as a source of explants were: I. alba Reinw ex Blume,E. camaldulensis Dehnh, I. grandis Hill ex Maiden, I. robusta Sm.,E. saligna Sm, and E. tereticornis Sm.

1. SterilizationAfter collection from the field or the greenhouse, the materialswere washed with commercial detergent and rinsed in distilledwater. Following this, the materials were taken to the transferroom where they were soaked in an aqueous solution of 20 percent by volume of "Clorox" and sterile double distilled waterfor a period of 20 minutes. Next, the materials were rinsedtwice in sterile double distilled water and placed in a 0.01 'NHCl solution for 5 minutes. (This technique using O.OlN HClsolution was-:developed by Abdul-BakL(1974) who reported that theO.OlN HCl solution could wash away traces of hypochlorite remaining on

98

the surface of the material). Following the use of the HC1, thematerials were again rinsed twice in sterile double distilledwater. After these last two rinses, the materials were segmentedin sterile double distilled water.

2. Explant preparationSee Table 3 for an explanation of organs and parts of organsused and their rnethods of preparation.

Several media were used in the experiments. Composition ofdifferent media are shown as follows: Murashige and Skoog (1966) -Table 4; White (1963) - Table 5; linsmaier and Skoog (1965 - Table 6;Tulecke et ~., (1965) - Table 7; Nash and Davies (1972) - Table 8;Sommer et ~., (1975) - Table 9.

Macroelements stock solutions were made up separately, 50 xconcentrated for each salt, and mixed before use. Microelements stocksolutions were made up in a single stock solution, 50 x concentrated,for each media.

The growth factors solutions were prepared fresh and added tothe mineral salt solution before adjusting the pH. Sucrose and agarwere added to the medium after adjusting the pH.

The pH of all the media was adjusted to 5.5 with HCl or KOHsolutions prior to sterilization. In cases where coconut milk was used,CCM was added before the adjustment of pH.

Organs or partsof organs used

Sterile pieees of stem were exeised into 2 em lengthseither eontaining or not eontaining a node in theupper partoPetioles from adult fully developed leaves wereexeised into 1 em pieeesFully developed juvenile and adult leafblades wereexeised into 0.5 em side squaresApieal shoots were eut 2 em in length and lowerleaves were removedSterile flower buds were disseeted, anthers separatedfrom filaments, with only the anthers being used.The flower buds used were all at the same stage ofdevelopment. During this specifie operation, the useof metal instruments was avoided.Root segments 1 em in length were eut from the rootsystem of seedlings grown under sterile eonditions.Hypocotyl segments 1 em in length were excised fromseedlings grown under sterile eonditionsCotyledons of seedlings grown under sterile eondi-tions were removed and plaeed in the mediumAfter the sterilization proeedure, seeds were placedin the medi um without further treatment

NH4N03KN03.

CaC12·2H20MgS04·7H20KH2P04

Na2EDTA-~.

F~O'- ·7H o. ,·4 2

H3B03

MnSo4·4H20ZnS04·4H20KINa2Mo04·2H20CoC12·6H20

myo-inosito1-2.O rn9l1 Nicoti nic aci d1-30 rng/1 Pyri doxi n·HC1

0.04-10 rng/1 Thiarnin·HC1

rng/11650 -190044027017037.327.86.2

22.38.60.830.0250.025

10 g/l

100 rng/10.5 rng/10.5 rng/l0.1 rng/1

mg/1KC1 65KN03 80Ca(N03)2·4H20 300MgS04·7H2O 720Na2S04 200NaH2P04·H2O 16.5Fe2(S04)3 2.5MnS04·4H2O 7ZnS04·7H2O 3H3B03 1.5KI 0.75CuS04·5H2O 0.001Mo03 0.0001G1ycine 3.0Nicotinic acid 0.5Thiamine 0.1Pyridoxine 0.1

rng/1NH4N03 1650KN03 1900CaC12·2H2O 440MgS04·7H2O 370KH2P04 170Na2EDTA 37.3FeS04·7H2O 27.8H3B03 6.2MnS04.4H2O 22.3ZnS04·4H2O 8.6KI 0.83Na2Mo04·2H20 0.25CuS04·5H2O 0.025CoC12·6H20 0.025sucrose 30,000agar 10,000IAA (l-30)kinetin (0.001-10)thiarnine HC1 0.400rnyo-inosito1 100

mg/lMgSo4'7H2O 730.0Ca(N03)2'4H20 280.0Ha2S04 200.0KN03 80.0KC1 65-0NaH2P04·H2O 165.0H3B03 0.100MnS04·N2O 3.00ZnS04·7H2O 0.500NaMo04'2H20 0.025CuS04'5H2O 0.025Fe citrate 2.00Ca pantothenate 1.0Pyridoxine HC1 0.25Thiamin HC1 0.25Nicotinic acid 1.25G1ycine 7.50NAA 0.1Sucrose 20,000L-Arginine 210.0Agar 8,000.0

mgfl

MgS04'7H2O 250KCl 750NaN03 850CaC12'6H2O 110KH2P04 140H3B03 0.2MnS04·4H2O 1.0ZnS04'7H2O 0.5KI O.1CUS04'SH2O 0.02CoCL2'6H20 0.01Na2Mo04'2H20 0.02Ferric Citrate 5.0Ca pantothenate 1.0Pyridoxine HCl 0.5Thiamine HCl 5.0Inositol 100.02,4-D 1.105Kinetin 0.5Sucrose 20,000.0Agar 8,000.0

Na2EDTA 27.3 - was usedinstead of Ferric citrate

mgf1 mgf1

(NH4)2S04 200 MnS04H2O 10CaC12'2H2O 150 ZnS04·7H2O 3MgS04'7H2O 250 H3B03 3KN03 1000 CuS04·5H2O 9.25KC1 300 NaMo042H20 0.25KI 0.75 CoC12'6H20 0.25NaH2P04'H2O 90 Inosito1 10.0NaHP04 30 Thi amine HC1 1.0FeS04'7H2O 27.8 Nicoti nic Aci d 0.1Na2EDTA 37.3 Pyri doxi ne HC1 0.1Sucrose 20,000 NAA 2.0Agar 7,000 6-benzy1adenine 5.0

autoclave at 121° C with 15 lbs/sq. in. of pressure for a period of 20minutes. When preparing solid medium, 10 ml of the medium was placedin 30 ml French square bottles or 30 ml placed in 120 ml Frenchsquare bottles. When preparing liquid medium for the suspensionculture, 30 ml of liquid medium was placed in 250 ml Erlenmeyer flasks.When preparing medium used in the roller drum, 3 ml of media were

100 rpm in darkness at 28° C. Cultures grown on solidified agar mediawere kept in total darkness at 28° C, or in a photoperiodic conditionwith illumination of 5,000 lux for 12 hours from flourescent and

Àincandescent lamps and a temperature of 28° C and a 12 hour dark periodwith 23° C temperature.

In experiments where cell suspension culture was used, a smallpiece (~ 0.5 9 fresh weight) of friable callus was transferred to theliquid medium. When cells from cell suspension cultures were subcul-

Ce11 suspension cu1tures were pipetted in quantities of 2 m1on to the surface of solid media. The medium (30 m1 in 120 ml Frenchsquare bott1es) was previous1y solidified in the bott1es in horizontalposition.

Numerous experiments were made in order to se1ect a mediumfor cal1us induction and growth, and to achieve organogenesis orembryogenesis from the exp1ants, cal1us or cell suspension.

1. Se1ection of a medium for callus induction and growthSevera1 media (Murashige and Skoog, 1962; White, 1963;Linsmaier and Skoog, 1965; Tu1ecke et ~., 1965; and Nashand Davies, 1972) were used to estab1ish cu1ture ofEuca1yptus a1ba, Euca1yptus grandis and Euca1yptus sa1ignawith exp1ants from different parts of organs (leafb1ades,petio1e, stem, and apica1 shootfor a11 three species plusanthers on1y for Euca1yptus alba). These cu1tures weregrown under the photoperiodic condition cited.

2. Sterilization of the exp1antsBecause of the high percentage of contamination of cu1tures,(most1y by fungi) it was necessary to develop a bettersteri1ization procedure for the explant. Exp1ants fromjuveni1e and adu1t 1eafb1ades of Euca1yptus grandis growingunder greenhouse and fie1d conditions were used in Latin

Square (with 10 replieates) experiments where elorox eon-eentrations, 10-40% by volume, were tested for a periodof time from 10 to 40 minutes. The Nash and Davies (1972)medium was used for eultivating explants.

3. Anagonostakis (1974) suggested that the use of aetivatedehareoal in the medi um may remove some toxie substaneesfrom the explant or from the medi um. Aetivated ehareoal(Daero G-60; Matheson, Coleman and Bell) was added or notadded (8.0 g/l) to the medium before the sterilization; orsterile ehareoal added on the surfaee of the mediumduring the transfer of the explant. The Nash and Davies(1972) medium was used with or without the substitution of2,4-D by NAA in this experimento Explants from juvenile andadult parts of organs (leafblade, petiole and stem) ofEuealyptus grandis, Euealyptus robusta and Euealyptusteretieornis were used while only explants from leafbladesof juvenile Euealyptus saligna; anthers from flower budsof f. robusta with 2.0 and 2.5 em of length; and anthersfrom flower buds with 0.4 - 1.0 em of length, seeds, roots,hypoeotyl and eotyledon of seedlings of f. grandis wereused. All the treatments had 10 replieates and the eultureswere plaeed to grow under the photoperiodie eondition eited.

4. SueroseAmorin (1970) reported an inerease in eallus dry weightwith the inerease of suerose eoneentration in the mediumwith eultures of Paul's Searlet Rose. Explants from

1eafb1ades of adu1t Euca1yptus grandis were p1aced in Nashand Davies (1972) medium with sucrose concentrationsranging from O - 50 g/l. All treatments had 20 replicatesand were placed to grow under the photoperiodic conditiondescribed.

5. Amorin (personal communication) suggested the use of boronfor the control of phenolic production in tropical plants.Explants from leafblades of adult Eucalyptus grandis wereplaced on Nash and Davies (1972) medium with H3B03 concen-trations ranging from 0.2 - 102.4 mIl. All treatments had20 replicates and were cultured under the photoperiodicregime described.

6. NitrogenNumerous works have been done on nitrogen nutrition andmetabolism and its role on morphogenesis (Streeg, 1969 and1973; Amorin, 1970; and Sommer, 1972). Explants fromleafblade of adult Euca1yptus grandis were cu1tured in Nashand Davies (1972) medi um where the nitrate source (NaN03)concentration varied from O - 1.5X with or without theaddition of ammonium (NH4C1) in different concentrations(O - 800 mg/1). Each treatment with 10 rep1icates wereplaced to grow under dark and light conditions.

7. Euca1yptus grandis x auxins/kinetinMuch experimentation has been performed pertainint to thehormona1 contro1 (auxin-kineti~ of morphogenesis of tobaccoreported by Skoog and Mil1er (1957). The Nash and Davies

(1972) medium was used as a basic medium and the concentra-tions of kinetin and 2,4-0 varied from O - 8.0 mg/l. When2,4-0 was substituted with IBA and NAA, their concentrationvaried from O to 8.0 mg/l and the kinetin concentrationfrom O to 1.0 mg/l. Because of the lAA -egradation(Hartman and Kester, 1968), it was used in concentrationsfrem 0.1 to 20 mg/l and the kinetin concentrations werefrom O to 1.0 mg/l. Explants from leafb1ades of adu1tEuca1yptus grandis were used to estab1ish these cu1tures.The number of rep1icates was 20 when 2,4-0 was used and10 replicates for the other auxins. These cu1tures weregrown under the photoperiodic and darkness conditionsdescribed.

8. Sommer et al., (1975) and the Nash and Oavies (1972) mediaSommer et ~., (1975) reported the development of embryosof Pinus species. The Sommer et ~., (1975) No. 1 mediumwith the normal CaC12·2H20 concentration or ha1f of thisconcentration, and the Nash and Oavies (1972) medium wereused to cu1ture adult 1eafb1ade exp1ants or ca11us fromEucalyptus grandis seed1ing juveni1e or adu1tmateria1.Each treatment"wi th 10 rep1icates were grown under thedarkness and photoperiodic conditions described.

9. GA3 pre-treatmentA 100 mg/l GA3 solution was or was not used to soak the1eafb1ade exp1ants for a period of 2 hours at room tempera-ture before the steri1ization of the exp1ants. The species

E. robusta and I. saligna - adult trees only, andE. grandis juvenile and adult trees. The medium used wasthe Nash and Davies (1972) with the 2,4-D concentration of1.0 mg/l and tbe kinetin concentration of 0.5 mg/l; andin other cases, substitutionof the 2,4-D with IBA (1 mg/l)and no kinetin was used. Each treatment had 20 replicateswith 10 replicates cultured under darkness and 10 replica-

leafblades of adult I. grandis tn the dark or without light.Each treatment had 10 replicates.

11. Coconut milk and hypocotylNash and Davies (1972) medium with 5,10 and 15% of CCM wasused to culture I. grandis hypocotyls under photoperiodicconditions. Each treatment had 10 replicates.

12. Cel1 suspension cultureCel1 suspension cultures were established from cal1us grownin Nash and Davies (1972) medium and transferred to liquidNash and Davies (1972) medium or to Sommer ét ~., (1975)

I. grandis material from seedlings. 1 year, 1 1/2 year and,

cell suspension cultures from explants in liquid media inthe roller drum and linear shaker. These were explantsfrom stem and leafblade with and without GA3 (100 mg/lfor 2 hrs.) pre-treatment.

13. PlatingCells from suspension culture of Sommer et ~., (1975) No.1 medium Nash and Davies (1972) medium were orginatedfrom callus of different ages of f. grandis trees. Thesecells were pipetted on Nash and Davies (1972): (1) normalmedium, (2) without 2,4-0 and knetin, (3) plus CCM, (4)plus CCM and 1 mg/l 2,4-0, (5) with 1 mg/l 2,4-0, (6)Sommer et ~., (1975) No. 1 medium, normal or (7) Sommeret ~., (1975) No. 1 medium with half the calcium concentra-

the photoperiodic condition.14. Nodal culture

culture of Eucalyptus grandis and success in regeneratingroots and the development of naked buds. Nodal segments,with and without GA3 (100 mg/l for 2 hrs.) pretreatment

~from adult f. alba, f. camaldulensis, f. robusta, f. salignaand adult and juvenile f. grandis were placed on basic Nashand Oavies (1972) medium with IBA (1 mg/l) devoid of kinetinand on the basic Nash and Davies (1972) medium with 2,4-0

(1.0 mg/l) plus kinetin (0.5 mg/l). The cultures weregrown under the dark and light conditions.

1. Callus CultureFresh and oven dry (110° C for 24 hrs.) weights wereobtained from the cultures established from explants.These weights were obtained by measuring the weight ofthe callus and explant and subtracting the average weightof the explant, in both cases fresh and oven dry weight.The fresh and oven dry weights referred to here areconsidered to be only the callus, per se. These evaluationswere made 60 days after the transferring of the explantson to the medi um.

The ratio between the fresh and over dry weight wasobtained in order to assist in the characterization of thecallus. Morphological description of the calluses was madebefore trabulating the callus fresh weight. Callus sub-cultures were only evaluatedby describing their morpholo-gical characteristics.

2. Cell suspension cultureCell suspensions were cultured for a 21 day period with thepurpose of being to produce material for cell plating. Nocytological studies of the cell suspension cultures weremade.

3. PlatingCultures established from cell suspensions on the surfaceof solid media (plating) were tabulated according to theculture growth and morphological characteristics of thecell "colonies" growth with a 60 day continuing period.

Nodal cultures were evaluated according to the root and/orbud development. If only callus was produced, its morpho-

Severa1 media (Murashige and Skoog, 1962; White, 1963; Linsmaierand Skoog, 1965; Tu1ecke et ~., 1965, and Nash and Davies, 1972) wereused to estab1ish cu1tures of Euca1yptus a1ba, Euca1yptus grandis andEuca1yptus sa1igna with exp1ants from different parts of organs (leafb1ades, petio1e, stem and apica1 shoot for a11 three species p1usanothers on1y for I. alba).

Exp1ants of 1eafb1ade of the three Euca1yptus species used werethe most usefu1 for ca11us production. Stem, petio1e and apica1 shootexp1ants, in that order, produced sma11 ca11uses which developed a brownhalo in the medium which inhibited ca11us growth. This browning of themedium or of the ca11us has been referred to as "pheno1ic oxidation" bytissue cu1ture workers (de Fossard, 1974; and Monaco et ~., In Press).The brown co1or in the medium or in the ca11us was not present in testsconducted with Euca1yptus a1ba anther exp1ants. The medium used inthese instances was Nash and Davies (1972) and the period of the test was6 months with three subcu1tures.

The Nash and Davies (1972) medium was se1ected in the experi-ments for ca11us induction and growth. This medium produced largerquantities of friab1e cal1us growth than other media tested.

In experiments, a high percentage of cu1ture contamination(most1y by fungi) was observed. A C1orox solution (15% by volume) wasused for a time period of 15 minutes for the steri1ization of exp1antsfrom trees growing under either fie1d or greenhouse conditions.Material from trees grown under fie1d conditions had a higher percentageof contamination than that frem trees growing under greenhouse condi-tions. Because of this prob1em, it was necessary to make some changesin the use of Clorox. Therefore an experiment was designed varying theC1orox concentration (10, 20, 30, and 40% by volume) and also varyingthe exposure period for the material being disinfected (10, 20, 30, and40 minutes).

Explants from the leafblade of Eucalyptus grandis were used inthis experiment because of their availability. The results of theexperiment are shown in Table 10. The use of Clorox, 20% by volume for20 minutes, was the most successful of all combinations tested. Adecrease in the Clorox concentration brought an increase in the percent-age of contamination while an increase both in the exposure period andClorox concentration inhibited ca11us growth or the explant became brownand subsequently died.

De Fossard (1974) and Creswe11 and de Fossard (1974) used adifferent procedure for steri1ization of Euca1yptus species materia1s,but the percentage of contamination was considerab1y higher, especia11ywhen material frem the fie1d was used.

Table 10: Results of Clorox treatment used in sterilization of Eucalyptus grandis leafblade explants.

Clorox 'Source Period of Time (minutes)Concentration of(% by volume) materials 10 20 30 40

100% cal1us growth 60% ca11us growth 50% ca11us growth 50% ca11us growthfie1d 80% contamination 60% contamination 50% contamination 20% contamination

10greenhouse 100% ca11us growth 60% cal1us growth 50% ca11us growth 50% ca11us growth

50% contamination 40% contamination 30% contamination 10% contaminationfie1d 60% ca11us growth 80% ca11us growth 60% ca11us growth 40% ca11us growth

30% contamination 20% contamination 10% contamination 10% contamination20 30% dead explants 50% dead explants

greenhouse 80% ca11us growth 100% cal1us growth 70% ca11us growth 20% ca11us growth20% contamination no contamination no contamination no contamination

30% dead exp1ants 80% dead explantsfie1d 70% ca11us growth 60% ca11us growth 50% cal1us growth

20% contamination 10% contamination no contamination 'no contamination10% dead exp1ants 40% dead exp1ants 60% dead exp1ants 100% dead explants

30greenhouse 70% cal1us growth 60% callus growth 50% ca11us growth

10% contamination no contamination no contamination no contamination20% dead explants 40% dead exp1ants 60% dead explants 100% dead exp1ants

field 20% cal1us growth 10% cal1us growth10% contamination 10% contamination no contamination no contamination

40 90% dead exp1ants 70% dead explants 90% dead exp1ants 100% dead explants-'-'CX>

CloroxConcentration(% by volume)

Sourceof

materials

40 greenhouse No contamination no contamination no contamination no contamination100% dead explants 100 % dead explants 100% dead explants 100% dead explants

Anagonostakis (1974) suggested that the use of aetivatedchareoa1 in the medium may remove some toxie substanees from theexp1ants or from the medi um. Aetivated ehareoa1 (Dareo G-60, Matheson,Co1eman and Se11) was added or not added to the medium (8.0 g/l) priorto steri1ization; or steri1e ehareoa1 was added on the surfaee of themedium during the transfer of exp1ants. The Nash and Davies (1972)medium was used with or without the substitution of 2,4-D by NAA.Exp1ants from juveni1e and adu1t parts of organs (leafb1ade, petio1e andstem) of Euea1yptus grandis, Euea1yptus robusta, and Euea1yptus tereti-cornis were used whi1e on1y exp1ants from leaf b1ades of juvenileEuealyptus saligna; anthers from flower buds with 0.0 - 1.0 em of length,seeds, roots, hypoeotyl and eotyledon of seedlings of I~grandis; andanthers from flower buds with 2.0 and 2.5 em of length of E. robustawere used to establish eultures.

All the treatments had 10 rep1ieates and the eu1tures weregrown under 12 hour photoperiod eonditions. Some friable eallus eu1turesof E. grandis were transferred to the media with eharcoal.

The explants originating from different parts of organs of thefour speeies used in these tests died or did not develop callus onculture media eontaining ehareoal or having ehareoal on the surface.Euealyptus grandis seeds normally deve10ped into normal seedlings on bothmedia containing chareoal while in the medium without ehareoal, the seedsdeveloped a teratoma-like callus with roots and shoots.

Pale yellow friable calluses of Eucalyptus grandis were trans-ferred to the medium with charcoal added prior to sterilization. Thegrowth of these callus cultures was inhibited when transferred to themedium without charcoal. Furthermore, the coloration changed from paleyellow, friable calluses to dark brown.

The concentration of sucrose in the nutrient medium's was usedto study the effects of carbohydrate concentrations on callus growthand root morphogenesis. Amorin (1970) reported an increase in thecallus dry weight with the increase of sucrose concentration in thenutrient medium. Hartman and Kester (1968) discussed the effects ofcarbohydrate content in the plant material used for vegetative propaga-tion on root morphogenesis. A high carbohydrate content seems to favorroot morphogenesis and inhibits shoot development.

Explants from leafblade of adult Eucalyptus grandis were culturedin the Nash and Davies (1972) mediumwith sucrose concentrations rangingfrom O - 50.0 g/l under 12 hour photoperiod conditions. Fresh and dryweights of the calluses increased with the increasing concentrations ofsucrose in the medi um. However, the ratio obtained between fresh/dryweights decreased with the increase in sucrose concentrations. Thefriability of callus cültures increased following an increase in thesucrose concentration in the culture medi um. At the lower sucroseconcentrations (O - 5.0 g/l) the calluses were dark brown and compactwhile at higher concentrations, they were greenish and friab1e. Character-istics of ca1lus growth at different sucrose concentrations are listed inTab1e 11.

Tab1e 11: Fresh and dry weights (mg) and the ratio between fresh and dryweights of cal1uses from leafblade exp1ants of Eucalyptusgrandis cu1tured in Nash and Davies (1972) medium with differ-ent sucrose concentrations under 12 hour photoperiod.

Sucrose weightmg/1 fresh

(mg)dry

ratiofresh/dry

0.0 14.7 2.41 6.10 dark brown compact ca11us aroundthe exp1ant

5.0 441.0 12.2 36.0 dark brown compact ca11us underthe exp1ant

10.0 960.6 26.2 36.7 brown-green friab1e ca11us underand around the exp1ant

15.0 1083. 1 35.3 30.7 pa1e green friab1e ca11us20.0 1275,0 52.7 24.2 pa1e green-white friab1e ca11us25.0 2008.0 79.8 25.2 pa1e green-white friab1e callus-

roots30.O 1569.O 79.5 19.7 pa1e green-white friab1e callus-

roots35.0 1242.5 85.7 14.5 pa1e green-white friab1e ca11us-

roots40.0 1727.5 110.0 15.7 pa1e green-white friab1e ca11us-

roots50.0 1170.4 87.0 13.5 pale green friable ca11us

Root morphogenesis oeeurred in the treatments with 25.0, 30.0,and 40.0 g/l of suerose. The roots were mostly aerial with hairs andhad approximate1y 2.0 em of 1ength (See Figure 11). Morphogenesis ofthe roots fo110wed the pattern diseussed by Hartman and Kester (1968)due to the faet that at the highest suerose eoneentration (50.0 g/l) inthe nutrient medi um, roots did not deve10p.

Inerease in the dry weight with the inerease in the sueroseconcentration in the medium (Amorin, 1970) occurred, but the increase inthe fresh weight and friabi1ity of the ca11uses was not expected due tothe fact that Amorin (1970) a1so reported an increase in the po1ypheno1of tissues cu1tured at higher sucrose concentrations. This wou1d beexpected to increase pheno1ic oxidation and thus inhibit ca11us growth.

Exp1ants from 1eafb1ades of adu1t Euca1yptus grandis were cu1turedin the Nash and Davies (1972) medium with the H3B03 coneentrations(0.2, 0.4, 0.8, 1.6, 3.2, 4.0, 12,8, 25.6, 51.2 and 102.4 mg/1). Eachtreatment was rep1icated 20 times and cu1tured under a 12 hour photo-period.

Fresh and dry weights of ca11uses and their characteristics are1isted in Table 12. Fresh and dry weights of cultures do not corre1ateto the increasing H3B03 concentrations. Most of the ca11uses were pa1egreen and friable. At 10wer concentrations (0.2 - 0.8 mg/1) cal1us wereproduced around the exp1ant, whi1e from 1.6 - 51.2 mg/1 of H3B03, theca11us grew on1y on the two opposite sides of the exp1ant (See Figure12,A). At the 102.4 mg/1 of H3B03, ca11us deve10ped on1y in the corners

Figure 11: Root morphogenesis in ca11us of Euca1yptus randis 1eafb1adeexp1ant cu1tured in the Nash and Davies (1972 mediumsupp1ied with 40 g/l of sucrose.

126

Tab1e 12: Ca11us characteristics, fresh and dry weights (mg) of ca11usesfrom 1eafb1ade exp1ants of adu1t Euca1yptus grandis cu1turedin the Nash and Davies (1972) medium and supp1ied withdifferent H3B03 concentrations. Eva1uation at the 60th day ofcu1ture in 12 hour photoperiod.

H3B03 weight ratio ca11us characteristicsmg/l Fresh oven dry fresh/dry

0.2 1809. O 62.5 28.9 pa1e green ca11us growth underthe exp1ant

0.4 1983.0 65.3 30.4 pa1e green ca11us growth aroundthe exp1ant

0.8 1710. O 60.8 28.1 pa1e green ca11us growth aroundthe exp1ant

1.6 1624.0 59.3 27.4 pa1e green ca11us growth onopposite side of exp1ant

3.2 1428.0 54.2 26.3 pa1e green ca11us growth onopposite side of exp1ant-roots

6.4 1709.0 57.4 29.8 pa1e green ca11us growth onopposite side of exp1ant-roots

12.8 1408.0 46.9 30.0 pa1e green ca11us growth onopposite sides of exp1ant-roots

25.6 1963. O 63.7 30.8 pa1e green ca11us growth onopposite sides of exp1ant-roots

51.2 1242.0 49.2 25.2 pa1e green ca11us growth onopposite sides of exp1ant

102.4 1119.0 30.6 36.6 brown pale callus growth in onlythe corners of th'~ exp 1ant

of ~he explant and grew towards the medium (See Figure 12,B). Observingthe habit of callus growth in this experiment, it seems apparent thatH3B03 concentration regulates the polarity of cell division and subse-quent callus growth. This is documented by the growth of callus (Table13). Root morphogenesis occurred in the treatments of 3.6, 6.4, 12.8and 25.6 mg/l of H3B03 concentrations. The roots were aerial and hadhairs (see Figure 13).

Amorin (personal communication) suggested the use of boron forthe control of phenolic production in tropical plants. However, Skok(1968) reported the synergistic effect of boron on the gibberellic acidinduction of stem swellings that involves intense proliferation of pri-

amary xylum tis sue in debudded tobacco plants. Bachelard (1969) reported~ .

the effects of gibberel1ic acid on internode growth and starch contentsof Eucalyptus camaldulensis seedlings. It was found that an activecenter of cell division was necessary for the GA3 to induce transversecell division and the carbohydrate content increased with the priximityof the active cell division center. Hartman and Kester (1968) reported

Explants from leafb1ade of adu1t Eucalyptus grandis werecultured in the Nash and Davies (1972) medium where tne nitrate source(NaN03) concentration varied from O - 1.5x with ar without the additionof NH4Cl in different concentrations (o - 800 mg/l). These cultureswere grown in the dark or in the 12 hour photoperiod. Fresh and dry

Ca11us growth habit in Euca1fptus grandis 1eafb1ade exp1antsin the Nash and Davies (1972 medium with: (a) 1.6 - 51.2mg/1, and (b) 102.4 mg/1 of H30H3 concentrations. Ca11usgrown under 12 hour photoperiod.

Figure 13: Roots from exp1ants of 1eafb1ade of adu1t Euca1yptus grandis,cu1tured in the Nash and Davies (1972) medium, supp1ied with3.2, 6.4, 12,8 and 25.6 mg/1 of H3B03. Cu1ture grown underthe 12 hour photoperiod.

observed in any of the treatments at the 60th day of culture. Theca11uses in both light and dark environments were pale brown, globulousand friable (See Figure 14).

an increase in the friability of the callus with the increase of nitrogenin the medi um.

(1969), Amorin (1970) and Sommer (1970) reported nitrogen nutrition andmetabolism in tissue culture. Hartman and Kester (1968) reported thathigh nitrogen content in the propogating material induces shoot develop-ment and inhibition of root development.

possible hormonal control of morphogenesis in the cultures establishedfrom explants of leafblade of adult I. grandis. The test of lAA (0.1,1.0, 5.0, 10.0 and 20.0 mg/l) and kinetin (0.0, 0.5 and 1.0 mg/l) incombinations produced roots only at 0.1 mg/l IAA and 20.0 mg/l of lAAand 1.0 mg/l kinetin. In the other combinations only calluses wereproduced. Fresh and dry weights are shown on Table 14.

'-C-"--'''''''''ll!I!

Tab1e 13: Fresh and dry weights (mg) of ca11us from adu1t Euca1yptus grandis 1eafb1ade exp1ants. Cu1turedin severa1 combinations of nitrate and ammonia supp1ied to the Nash and Davies (1972) madium .

NH4(mM) ""N03(mM) 0.0 0.5 2.5 7.5 151ight dark 1ight dark 1ight dark 1ight dark 1ight dark

FW 15.4 486.4 804.4 1019.O 1180.4 1206.4 1023.9 1190.4 1133.80.0. DW 1.6 30.9 58.9 49.9 51:6 48.3 45.4 45.6 47.2

Ratio 9.63 15.74 13.66 20.43 22.88 24.98 22.55 26.10 24.02FW 156.4 169.4 145.9 1534.4 1043.4 1201 .4

0.5 DW 7.6 6.6 20.6 57.1 41.6 55.6Ratio 20.58 25.67 7.08 26.87 25.08 21 .61FW 197.4 90.4 166.4 111.4 229.4 302.4 904.4 596.7 1244.4 1337.4

2.5 DW 11.6 1.6 6.6 7.6 8.6 15.9 29.6 27.2 38.1 10.6Ratio 17.02 56.5 25.22 22.55 26.67 19.02 30.55 21;94 32.66 126.17

FW 155.4 282.4 240.4 555.4 299.4 1349.4 981.0 1593.47.5 DW 7.6 12.9 8.6 30.1· 12.1 48.6 32.1 51.1

Rati o 20.45 21.89 27.95 18.45 27.74 26.77 30.57 41.18FW 404.4 245.41 187.4 569.4 560.4 386.4 1569.4

15.0 DW 12.6 6.1 2.6 19.6 24.1 16.6 39.6Ratio 32.09 40.23 72.08 29.05 23.23 23.23 39.63

Ca11us from 1eafb1ade exp1ants of adu1t Euca1ãptus grandis atthe 60th day of culture in 12 hour photoperiousingdifferent nitrogen sources and concentrations.

Tab1e 14: Fresh and dry weights (mg) for ca11uses from exp1ants of1eafblades of adu1t Euca1yptus grandis~ Cu1ture grown in12 hours photoperiod for 60 days. lAA and Kinetin.

Kinetin (mg/1)~AA (mg/1) O.1 1.0 5.0 10.O 20.0

FW 8.7 37.7 87.7 28.2 98.00.0 DW 0.4 0.6 1.1 0.3 2.2

Ratio 21.75 62.83 79.9 94.0 44.54FW 114.2 254.0 571.4 1216.7 1791.4

0.5 DW 11.8 21.0 53.1 58.6 98.9Ratio 9.68 12.10 10.76 20.76 18.11FW 151.4 91.4 533.6 1318.7 1395.61.0 DW 10.2 8.9 39.8 69.6 64.8

Ratio 14.84 10.27 13.41 20.05 21.54

Combinations of IBA and NAA concentrations (0.01,0.5,1.0,2.0,4.0 and 8.0 mg/1) with kinetin concentrations (0.0,0.5 and 1.0 mg/1)were used with exp1ants of 1eafb1ade of adu1t ~. grandis and cu1tured in12 hour photoperiod. Root morphogenesis occurred in the combinationsof IBA and kinetin. The best root growthwas obtained at 1.0 mg/1 ofIBA and no kinetin (See Figure 15). The fresh and dry weight of thecu1tures are 1isted in Tab1e 15.

In the NAA test, roots were produced on1y in the experiments:4.0 and 8.0 mg/1 of NAA and no kinetin; 1.0, 2.0, 4.0, and 8.0 mg/1 ofNAA p1us 0.5 mg/1 kinetin; and 2.0,4.0 and 8.0 mg/1 of NAA p1us 1.0rng/1 of kinetin. Fresh and dry weights of the cu1tures are shown inTab1e 16.

In experiments using 2,4-0 and kinetin at concentrations of(0.0,0.5, 1.0,2.0,4.0 and 8.0 mg/1 in combination, roots were pro-duced in the fo110wing cases: 2.0, 4.0, and 8.0 mg/1 of 2,4-0 a10ne;0.4, 1.0, 2.0, 4.0 and 8.0 mg/1 of kinetin a10ne and in the combinations:4.0 mg/1 2,4-0 p1us 1.0 mg/1 kinetin, 8.0 mg/1 2,4-0 p1us 1.0 mg/1kinetin, 1.0, 4.0 and 6.0 mg/1 of 2,4-0 p1us 4.0 mg/1 of kinetin, andin the combination of 0.5 mg/l of 2,4-0 plus 6.0 mg/l of kinetin. Most ofthe roots produced were aeria1, even in the same ca11us with one or tworoots growing in the medium and the other 6 to 10 roots projecting intothe air. Fresh and dry weights of the cultures are in Table 17.

In experiments with 10wer 2,4-0 concentrations (0.01 and 0.1rng/1) and kinetin (0.0, 0.5 and 1.0 mg/l) cultured in both dark and1ight, root deve10pment occurred in 1.0 mg/l kinetin and 0.0 mg/l of2,4-D in the light whi1e the experiments in the dark did not develop

Figure 15: Root deve10prnent in exp1ants of 1eafh1ades of adu1tEuca1yptus grandis in the Nash and Davies (1972) mediurnsupp1ied with 1.0 rng/1 of IBA and 0.0 rng/1 of kinetin.Cu1tured in 12 hour photoperiod.

Tab1e 15; Fresh and dry weights (mg) of ca11uses from exp1ants of 1eaf-b1ade of adu1t Euca1yptus grandis. Cu1tures grown in 12 hourphotoperiod for 60 days. IBA and Kinetin.

kinetin (mg/1~A (mg/1) 0.01 0.5 1.0 2.0 4.0 8.0

FW 88.4 2.4 94.4 54.10 781.0 467.70.0 DW 0.9 0.2 0.8 42.4 42.8 33.1

Ratio 98.22 12.0 118.0 12.76 18.25 14.13

FW 517.8 850.6 1148.2 1115.2 968.2 1792.40.5 DW 43.6 10.8 69.0 88.4 56.4 88.6

Ratio 11.88 10.8 16.64 12.62 17.17 20.23

FW 10.1 465.2 981.0 848.2 870.7 129.61.0 DW 6.1 41.4 70.8 60.6 65.4 69.2Ratio 1.66 11.24 13.86 14.0 13.31 1.87

Tab1e 16: Fresh and dry weights (mg) of ca11uses from exp1ants of 1eaf-b1ade of adu1t I. grandis. Cu1tures grown in 12 hour photoperiod for 60 days. NAA and Kinetin.

Kinetin (mg/1~AA (mg/1) 0.01 0.5 1.0 2.0 4.0 8.0

FW 7.4 76.0 219.2 49.4 529.80.0 DW 1.1 6.8 14.2 3.8 32.8

Ratio 67.2 11.18 15.44 13.0 16.15

FW 1766.4 634.0 685.0 989.0 1060.2 1177.60.5 DW 21.8 26.2 48.0 66.8 68.8 68.2

Ratio 81.03 24.24 14.27 14.81 15.41 17.27

FW 180.2 498.0 380.6 449.8 707.2 926.61.0 DW 20.0 43.2 39.4 47.6 66.8 73.2Ratio 8.66 11.53 9.66 9.45 10.59 12.66

Tab1e 17: Fresh and dry weights (mg) of cu1tures of exp1ants of 1eafb1ades of Euca1yptu~gran~ growingin 12 hour photoperiod.2,4-D and Kinetin.

Kinetin (mg/1~2,4-D (mg/1) 0.0 0.5 1.0 2.0 4.0 6.0 8.0

FW 178.4 173.0 438.0 156.5 691.O - 878.00.0 DW 13.4 18.4 35.2 10.4 40.8 - 46.2

Ratio 13.31 9.40 12.47 15.04 16.95 - 19.OFW 898.8 1545.4 1591.4 1620.4 1652.4 1853.6 1524.0

0.5 DW 45.4 59.8 47.4 67.0 65.8 78.0 q3.0Ratio 19.80 25.84 33.57 24.19 25.11 23.76 24.19FW 832.9 1783.8 1614.4 1416.4 1278.4 1743.4 1365.4

1.0 DW 39.1 56.2 55.0 56.8 53.2 56.8 56.6Ratio 21.30 33.91 29.84 24.93 24.03 30.69 24.12FW 1396.8 1297.6 1516.6 2017.8 1521;2 1551.6 3737.4

2.0 DW 45.6 44.8 59.0 66.0 51.4 48.2 118.2Ratio 30.63 28.96 85.71 30.57 29.60 32.19 31.62FW 1209.0 1269.0 1197.8 2340.4 1237.8 1530.8 1318.4

4.0 DW 42.8 42.2 50.2 54.6 44.4 50.6 35.8Ratio 28.25 30.07 23.86 42.86 27.88 30.25 36.83FW 1721.8 816.6 1706.2 1337.0 1396.2 2579.4 962.4

6.0 DW 43.6 34.2 57.0 50.6 30.2 53.2 45.6Ratio 35.42 23.88 29.93 26.42 46.23 48.48 21.11

FW 1418.4 955.4 1064.4 1694.4 1588.4 1842.4 1552.68.0 DW 43.6 37.8 36.2 48.2 49.2 51.2 55.0

Ratio 32.53 25.40 29.40 35.15 32.28 35.98 28.23--'~~

roots. However, the ca11us habit growth was compact and globu1ar (SeeFigure 16). The fresh and dry weights of the ca11us grown in the darkand in the 1ight are shown in Tab1e 18.

Marcavi11aca and Monta1di (1964), obtained root morphogenesis instem exp1ants of Euca1yptus trabuti with a very high concentration ofIAA (100 mgj1), and in experiments with various combinations of kinetinand IAA, morphogenesis was not observed. De Fossard (1974) reportedroot morphogenesis in undifferentiated ca11us of ~. grandis seed1ingswith 2,4-0 or lAA. However, Lee and de Fossard (1974) fai1ed to obtainregeneration of organs with various auxins (IAA, NAA., 2,4-0 and NOA)and cytokinins (kinetin and BAP) in cu1ture of stem and 1ignotuber ofE. bancrofti. De Fossard et ~., (1974) were not successfu1 in obtain-ing morphogenesis in cu1tures of severa1 Euca1yptus species in experi-~nts with various concentrations of cytokinins and auxins, or even inmixed cu1tures of Euca1yptus ca11us and regenerating tobacco ca11us.

A11 the 1eafb1ade exp1ants in both Sommer et ~., (1975) No. 1and ha1f ca1cium media produced pa1e white, ye11ow, greenish and redfriab1e ca11uses with very slow growth in the 1ight. Under darkconditions, the ca11uses were friab1e and pa1e ye110w or white with slowgrowth during the first six weeks. Afterwards, the cu1tures became fastgrowing. When ca11uses from Nash and Oavies (1972) medium weresubcu1tured in the Sommer et ~., (1975) No. 1 medium and grown indarkness, they grew slow1y during the first 8 week cu1ture period. The

Figure 16: Callus from explants of leafblade of adult Eucalyptus grandisin the Nash and Davies (1972) medium with 1.0 mg/l kinetinplus 0.1 mg/l of 2,4-D. Culture grown in darkness

Táb1e 18: Fresh and dry weight (mg) of cu1tures of 1eafb1ade exp1antsof adu1t Euca1yptus grandis in Nash and Davies (1972) mediumunder different 1ight conditions.

kinetin (mg/1) "'2,4-D (mg/1) Light Dark0.01 0.1 0.01 0.1

FW 94.9 587.9 79.4 490.60.0 DW 29.1 38.1 8.6 43.4

Ratio 3.26 15.43 9.23 11.30

FW 246.4 888.4 166.9 772.O0.5 DW 24.6 80.6 26.6 86.1

Rãtio 10.02 11.02 6.27 8.97

FW 125.4 753.4 178.4 785.71.0 DW 11.6 48.6 25.1 102.7Ratio 10.8 15.50 7.11 7.64

ca11~s was pa1e white, compact, and globular ,with a resemb1ance toear1y stages of embryogenesis (See Figure 17). If the callus fromNash and Davies (1972) medium was subcultured in the Sommer et ~.,(1975) No. 1 medium under a 12 hour photoperiod, it deve10ped ye110w,white, green and red coloration and was very slow growing unti1 the 6thweek of cu1ture. The fresh and dry weights of ca11uses from explantsof leafb1ade of adu1t Euca1yptus grandis are listed in Table 19. It canbe noted that there was a decrease in cal1us weight when grown in thedark and the ca11us dry weight increased while under a 12 hour photo-period resulting in a cal1us with a higher fresh weight.

Anatomica1 studies were not made in cal1uses subcultured in theSommer et ~., (1975) medium, but the callus structure and morphologicalcharacteristics were the same as described by Sommer et ~., (1975) andreported as embryos.

Kochba et ~., (1974) used gibberellic acid for the stimulationof rooting of Citrus embryoids.

Root morphogenesis occurred in al1 the cultures with or withoutthe GA3 pre-treatment of the leafblade explants of the species used inthis investigation: Eucalyptus alba, I. camaldulensis, I. robusta andI. saligna adu1t trees and I. grandis adult and juvenile trees in theNash and Davies (1972) medium with 0.1 mg/l of IBA substituting the2,4-D and no kinetin. The explants of Eucalyptus robusta with the GA3pre-treatment produced shoots (See Figure 18). The fresh and dry weights

'igure 17: Ca11us from the Nash and Davies (1972) medium subcu1turedin the Sommer et a1., (1975) No. 1 medium in the dark afterthe 8th week or-cUlture.

'~·'.1'r"~~;!tr,. ,~,~

~------------------------------------

Tab1e 19: Fresh and dry weight (mg) of ca11us from adu1t Euca1yptusgrandis leafblade explants

Medium Composition Light Dark

l. Nash and Davies (1972) FW 57.0 78.4no 2,4-D, no kinetin DW 1.0 1.4

Ratio 57.0 56.02: Nash and Davies (1972) FW 2294.1 1715.1

1 p1us CCM (15%) DW 95.9 76.8Ratio 23.9 22.33

3. Nach and Davies (1972) FW 423.5 156.32 p1us 2,4-D (1.0 mg/1) DW 47.7 12.2

Ratio 8.88 12.814. Sonmer et ~. , (1975) FW 1222.5 434.0

DW 59.6 28.2 .Ratio 20.50 15.39

Figure 18: Roots and shoots (arrows) morphogenesis in explant ofleafblade of adult Eucalyptus robusta with GA3 pretreatmentand cultured in the Nash and Davies (1972) medium with 1.0mg/l IBA and 0.0 kinetin. Cultures grown under 12 hourphotoperiod.

are shown in Table 20. The fresh and dry weights did not change con-siderably due to the fact that most of the growth in the cultures waslimited to roots.

The pre-treatment of the explants with GA3 did not induce rootmorphogenesis in the cultures of explants of leafblades in the Nash andDavies (1972) medium with 1.0 mg/l of 2,4-0 and 0.5 mg/l of kinetin.Calluses in all treatments were yellow-pale, both in the dark and in thelight. Fresh and dry weights of these cultures' are shown in Table 19.The GA3 pre-treatment increased the fresh weight and dry weight of thecallus cultured in the dark. In the light, however, only the callusesfrom Eucalyptus grandis juvenile material had a significant increase infresh and dry weight. There is no previous citations in the literatureon the use of GA3 and its effect on Eucalyptus tissue culture.

The experiment with the CCM to establish culture of explants ofleafblade of adult Eucalyptus grandis in the dark and in the light didnot show morphogenesis, but the culture in the media with CCM (15%) and2,4-0 (1.0 mg/l) in the light ar in the dark had an interesting patternof callus growth which can be characterized by brown coloration,compactness, and globular structural patterns resembling the early stagesof embryogenesis (See Figure 19). The media with only CCM (15%) producedyellow-white friable callus with hairs on the surface. The fresh anddry weights of cultures are shown in Table 19. The fresh weight of thecultures in the CCM (15%) alone decreased in the dark.

Table 20: Fresh and dry weights (mg) of calluses from explants of leafblade cultured in "the Nash and Davies(1972) medium with 1.0 mg/1 of IBA and 0.0 kinetin

Light DarkSpecies Without GA3 With GA3 pre-treatment Without GA3 With GA3 pre-treatment

Euca1yptus a1ba FW 436.9 495.4 420.4 310.9(adult) DW 42.6 42.1 45.6 35.6

Ratio 10.26 11.77 9.22 8.73Euca1yptus cama1du1ensis FW 259.9 148.4 74.9 134.4

(adult) DW 28.6 14.1 6.1 9.6Ratio 8.84 10.52 12.28 14.0

Euca1yptus grandi s. " I J FW 155.9 145.9 143.4 140.9

(adult) 1. DW 17.6 11.6 13.2 3.6Ratio 8.86 12.58 10.86 39.14

Euca1yptus grandis FW 132.4 72.9 628.9 424.4(adu1t) DW 16.1 9.6 58.6 42.1

Ratio 8.22 7.59 10.73 10.08Euca1yptus ro~usta FW 156.4 352.9 723,4 437.9

(adult) DW 16.6 37.1 59.1 44.1Ratio 9.42 9.51 12.24 9.93

Euca1yptus sa1igna FW 241.9 146.9 417.4 136.9(adult) DW 19.1 11.1 31.1 13.1

Ratio 12.66 13.23 13.34 10.45

Figure 19: Callus produced from exp1ant of leafb1ade of adult Eucalyptusgrandis cultured in the Nash and Oavies (1972) medi um,supplemented with 15% coconut mi1k p1us 1.0 mg/l of 2,4-0.

Euca1yptus ca11us can grow in media devoid of CCM. If CCM isadded to the medium, it was not necessary to add auxin (2,4-0). in the~dium for ca11us growth as reported by Jacquiot (1961) and Sussex(1964). However, the independence of Euca1yptus species ca11us from CCMwas reported before by Marcavi11aca and Monta1di (1964) and de Fossard~1-ª.l., (1972).

Hypocoty1 (1,0 segment) of Euca1yptus grandis seed1ings werecu1tured in the Nash and Oavies (1972) medium with or without coconutmi1k supp1ement added (5.0, 10.0 and 15%) and grown under a 12 hourphotoperiod. Regeneration of roots was inhibited by the exogenous CCM.Roots from the ca11using hypocoty1 exp1ant are shown in Figure 20. Thefresh and dry weight increased with an increase of CCM but the freshweights do not corre1ate we11 with CCM concentration (5% - 29.9 mg,10% - 49.9 mg and 15% - 34.9 mg). The ca11uses were pa1e-greehish-whiteand friab1e with hairs on the surface (See Tab1e 21).

Bache1ard and Stowe (1964) reported that CCM induced root growthin cu1tures of roots of I. cama1du1ensis, and that yeast extract had aninhibitory effect on root growth. In an experiment, root morphogenesiswas inhibited with the addition of CCM to the media in exp1ants that hadbeen producing roots in the same media devoid of CCM.

Ca11us of EUCa1Yytus grandis hypocoty1 cu1tured in the Nashand Davies (1972 medium under 12 hour photoperiod.

Tab1e 21: Fresh and dry weight (mg) of Euca1yptus grandis hypocoty1cu1ture with coconut mi1k under the 12 hour photoperiod.

WeightCCM%

fresh dry ratio

0.0 533.1 16.9 31.48

5.0 621.7 29.3 21.24

10.O 840.7 49.9 18.71

15.0 859.4 34.9 24.60

Cell suspension cultures were established from callus grown inthe Nash and Davies (1972) liquid medium and Sommer et ~., (1975) No. 1medium with the normal calcium concentration (1) or with half calciumconcentration (2). The calluses were from Eucalpytus grandis materialfrom seedlings (S), 1 year (J), 1 1/2 year (I), and adult (A) trees.These cultures were placed in either a tinear shaker or roller drum. Nomorphological analyses were made of the cells and cells aggregates becausethe objective of this experiment was to produce cells from platingexperiments. The cultures in the linear shaker developed a brown colorafter the 3rd week of culture, while in the roller drum they couldremain up to 10 weeks without browning in the culture. However, thecultures in the roller drum had a very low cell density.

The establishment of cell suspension cultures directly fromexplants (stem and leafblade), with or without GA3 pre-treatment in thethree media used, was possible only when cultured in the roller drum.Callus were initiated from cells in the periphery of explants and allover the surface of the stem segment explants.

The only work with Eucalyptus cell suspension culture wasreported by Sussex (1964). It was reported that cel1 growth was betterwhen the cultures were stabilized from large aggregates of cells, andwith small cell aggregates or single cell cultures grew slowly. Street(1973) discussed the nursing effects of cells in cultures or in the pre-conditioning of the medium by cell cultures.

Cells from cell suspension culture of Sommer et ~., (1975) No. 1medium with the normal calcium concentration (1) and half the calciumconcentration (2) and Nash and Oavies (1972) medium were originated fromcallus of different ages of Eucalyptus grandis tree seedlings (S), 1year old (J), 1 1/2 year old (I) and from adult (A) trees. These cellswere pipetted on Nash and Oavies (1972): ,(1) normal medium, (2) no2,4-0 and no kinetin, (3) 1 mg/l 2,4-0 and no kinetin, (4) CCM and no2,4-0 or kinetin, (5) CCM, 15% plus 1.0 mg/l of 2,4-0 and no kinetin,(6) Sommer et ~., (1975) No. 1 normal medium, and (7) Sommer et ~.,(1975) No. 1 medium with the normal calcium concentration. Thesecultures were grown under darkness or under a 12 hour photoperiod. Theresults are shown in Tables 22, 23, 24, and 25.' Root morphogenesisoccurred only in dark conditions in the cell suspension cultures ofseedling cal1us grown in the Sommer et ~., (1975) No. 1 medium withthe normal calcium concentration transferred to the Nash and Oavies(1972) medium with 1.0 mg/l of 2,4-0 and no kinetin; and from cell sus-pension from callus of 1 1/2 year old trees transferred to the Sommeret ~., (1975) medium normal. Examples of small globular cultures areshown in Figure 21.

Street (1973) reported on the use of conditioned medium forcell culture in plating experiments. In the experiments connected withthis research, cells were able to grow without the conditioning medi um.Perhaps this was because of the technique which was used: the cells werepipetted on to the surface of the medium with medium where the cells weregrowing previously.

510~1512520521522530531532540541542

550551552560561562

570571572

pa1e-white-red 1amina growthpa1e friab1e globu1espa1e friab1e globulespa1e-ye11ow compact sma11 globu1espa1e friab1e globu1espa1e friab1e 1arge globu1espa1e friab1e globu1es

Pa1e globu1espa1e friab1e globu1es-hairspa1e friab1e globu1es-hairspa1e compact globu1espa1e compact globu1espale compact globulesbrown friable sma1l globulespale brown compact globulespale brown small globulespale globules with smooth surfacepale (small) globu1es with smooth surfacepale red friable globules

yellow friable globulesyel10w friable globu1esyellow-white globulespale white small globulespale yellow globulespale yellow large globulesyellow small globulespale globules-rootsyellow friable globules

pale compact globulespale white compact globulespale white compact globules-roots

J10JllJ12

J20J21J22J30J31J32

J40J41J42

J50J51J52

J60J61J62

J70J71J72

yellow friable globulesyellow green friable globulesyellow friable globulespale friable globules - hairno growthpale small globules

yellow friable large globulespale brown friable globulesyellow friable globules-dropletspale small friable globulespale small compact globules

pale friable globulespale green globulespale globules

•..•..(J"l

___________________________ ---------- -aJ

pale red compact globulespale green globulespale green globulespale yellow red small globulespale compact globulespale small globulespale friable small globulespale friable small globulespale friable small globules

pale friable globulespale compact globulespale yellow compact globulespale friable small globules-dropletspale brown small globulesbrown small globules

110111112

120121122

130131132

140141142

150151152

160161162

170171172

Light Dark

pa1e friab1e globu1es yellow friab1e globu1espa1e friab1e globu1es ye110w friab1e globu1espa1e friab1e grobu1es ye110w sma11 globu1espa1e friab1e globu1es pa1e small globu1espa1e compact globu1es pa1e friab1e globu1espa1e compact globu1es pa1e friab1e globu1es

pa1e friab1e globu1espa1e white friab1e globu1es-hairsno visib1e ca11us growthpa1e friab1e globu1espa1e compact globu1espa1e friab1e globu1espa1e friab1e 1arge globu1espa1e sma11 friab1e globu1espa1e sma11 compact globu1espa1e green red friab1e globu1espa1e green red friab1e globu1espa1e green red friab1e globu1es

pa1e ye110w white friab1e globu1es-hairspa1e friab1e globu1espa1e compact globu1espa1e friab1e globu1es

pa1e compact globulespa1e compact globulesno ca11us growth

pale small compact globu1es

Table 24: Characteristics of plating cultures of Eucalyptus grandis adult (6 years)

AlOA11A12

A20A2lA22

A30A3lA32

A40A4lA42

A50A5lA52

A60A6lA62

A70A7lA72

Pale friable globulespale friable globulespale friable globulespale friable globulespale friable globulespale small friable globules

pale friable globulespale yel10w friable globulespale yel10w friable globules-hairspale yellow friable globulespale yellow friable globulespale yellow friable globules-hairspale red friable globulespale red friable globulespale red friable globulespale yellow red friable globulespale friable globulespale friable small globules

yellow friable globulesyellow friable globulesyellow friable globulespale white small globulespale small globules

pale compact globulespale friable globulespale compact globules-hairspale brown friable globulespale white compact globulespale white friable globulespale yel10w friable globulespale small friable globulesyellow campact globules-hairs

J•..~.. ~•••. .a. ~

••1 •. ._ _...--#.: . -~. - - - ; J

~.:,•..-.,'~

of Euca1yptus grandis and success in regenerating roots and the develop-ment ofaxi11ary buds.

Noda1 segments, with or without GA3 (100 mg/1 for 2 hours) pre-treatment form adu1t E. a1ba, ~. cama1du1ensis, I. robusta, I. salignaand adu1t and juveni1e E. grandis were p1aced on basic Nash and Oavies(1972) medium with IBA (1.0 mg/1) devoid of kinetin and on the Nash andOavies (1972) medium with 2,4-0 (1.0 mg/1) p1us kinetin (0.5 rng/1).

In the experirnent with the 2,4-0 and kinetin, root deve10pmentoccurred in the nodes of I. sa1igna with the GA3 pre-treatrnent in the1ight conditions; shoot deve10pment occurred in the nodes of I. cama1-du1ensis with the GA3 pre-treatment and grown in the 1ight. In othertreatments of this test, on1y ca11us growth occurred. The resu1ts ofthe test with IBA are shown in Tab1e 26. Shoots deve10ped from theinternode of E. cama1du1ensis without the GA3 pre-treatment grown in thedark (Seé Figure 22).

Naked bud deve10pment and no root deve10pment was observed inE. grandis juveni1e without the GA3 pre-treatment and grown in the1ight (See Figure 23), whi1e in I. grandis adu1t with the same treatment,roots and naked buds deve10ped (See Figure 24). Oeve10pment of roots,naked buds and adventitious buds was observed in E. robusta in bothtreatments in the dark (See Figure 25). Euca1yptus a1ba nodes did notdeve10p roots under any of the conditions in this experimento

Tab1e 26: Resu1ts of noda1 .(stem segment) cu1ture of Euca1pytus species in the Nash and Davies (1972)medium with IBA (1.0 mg/1) devoid of kinetin

Euca1yptus species No GA3 With GA3 No GA3 With GA3pre-treatment pre-treatment pre-treatment pre-treatment

E. a1ba (adu1t) ca11us growth, ca11us growth ca11 us and shoot no root orshoot deve10pment deve10pment shoot

deve10pmentE. cama1du1ensis (adu1t) root and shoot root deve1opment, shoot and root root deve1op-

deve10pment no shoots deve10pment ment, noshoot from inter- shootnode was observed

E. grandis (juveni1e) shoot deve1opment, root deve1opment, ca11us and shoot rootno roots no shoots deve1opment, no deve10pment

rootsE. grandis (adu1t) root and shoot root and shoot root and shoot root and shoot

deve10pment deve10pment deve10pment deve10pmentE. robusta (adu1t) rQot and shoot root and shoot root and shoot root and shoot

deve10pment deve10pment deve10pment deve10pmentE. saligna (adult_ ca11us growth, no ca11us growth, no root or shoot no root or

shoot or root no root or shoot deve10pment shootdeve10pment deve10pment deve10pment

--'"'-J

N

Shoot development from the internode of E. camaldulensis(adult tree) in the Nash and Davies (197l) medium with IBA(1.0 mg/1) devoid of kinetin, without the GA3 pre-treatmentand grown in the dark.

Figure 23: Deve10pment of naked bud of f. grandis juveni1e, without GA3pre-treatment, cü1tured in the Nash and Davies (1972) mediumwith IBA (1.0 mg/1) devoid of kinetin under 1ight conditions.

Figure 24: Deve10prnent of naked bud and roots of I. grandis adu1t, innoda1 cu1ture in the Nash and Davies (1972) with IBA (1.0rng/1) and devoid of kinetin. Cu1ture grown under 1ightconditions.

Figure 25: Development of naked and adventitious buds of E. robusta inthe Nash and Davies (1972) medium with IBA (1.0 mg/l) devoidof kinetin. Culture grown in the dark.

Creswe11 and de Fossard (1974) used 1iquid media without auxinfor noda1 cu1ture of Euca1yptus grandis, ~. bancroftii and I. degu1pta;microbia1 contamination was a 1imiting factor and the presence of leavesor segments of 1eaves was necessary.

In the experiments conducted in connection with this research,solid medium was used, and it was not necessary to have leaves or seg-ments of leaves in the nodes. In most of the cases, there was abscissionof the 1eaves or petioles in the first week of cu1ture.

It is possible to achieve diverse objectives through the tissueculture method of plant propogation. It is most often sought as analternate in the propogation of cultivars when conventional methodspermit only slow increasesin clonal plants. Tissue culture is parti-cularly helpful when used in conjunction with plant breeding programs.It enables the timely increase and hastens the availability of newvarieties. Even with cultivars that are propogated readily throughcuttings, divisions, and other conventional asexual techniques, thetissue culture method can be utilized to enhance substantially the rateof multiplication. A millionfold increase per year in the rate of clonalmultiplication over conventional methods is not unrealistic. Hence,with herbaceous plants that are already propogated quite rapidly by theconventional methods, tissue culture is a desirable aid in meetingspecial needs. In commercial nurseries, tissue culture can also be usedto minimize the growing space usual1y provided for the maintenance ofstock plants. Furthermore, when properly executed, the method can beused in the reproduction, improvement, and maintenance of relativelydisease free plants.

The use of tissue culture techniques for vegetative propogationand improvement of trees is highly desirable and has been used in severallaboratories to achieve a better method for tree propogation andimprovement. Much more has to be understood in the chemical composition

of the tree, mainly the phenolic compounds and their role in the controlof growth and development of the tree. It is well known that morpho-genesis of tree species does not follow the system developed by Millerand Skoog (1957) for tobacco, where changes in the auxin and cytokininconcentration in the media control the development of roots and shootsfrom the callus.

It would be untimely to suggest thattissue culture methods ofEucalyptus species,is near success; clearly, several technical problemsremain to be solved before this can be claimed since it is known thatchemical composition (phenolics) varies within the species.

Juvenility is yet another factor that has to be understood inwoody plants in order to achieve a better vegetative propogation throughthe conventional methods or tissue culture. Tissue culture, due to itsmethodology, would be suitable to learn more about juvenility. It isyet toe soon to assume that tissue culture techniques can resolve theproblems inherent within the conventional methods of vegetative propoga-tion of trees.

Explants from different parts of organs of six Eucalyptusspecies (I. alba~ I. camaldulensis~ I. grandis~ I. saligna~ I. robusta~and I. tereticornis) were used in all cell and tissue culture systemsto study the induction and growth of callus and morphogenesis.

Several media (Murashige and Skoog~ 1962; White, 1963; Linsmaierand Skoog~ 1965; Tulecke et ~., -1965; and Nash and Davies, 1972) wereused to establish cultures of E. alba~ I. grandis~ and I. saligna withexplants from different parts of organs (leafblade~ petiole~ stem andapical shoots of al1 three species in addition to anthers on1y for I.alba). The Nash and Davies (1972) medium was se1ected in the experimentsfor callus induction and growth. In this medium~ the grown callus waslarger and more friable when compared to the ca11uses grown in the othermedia.

The use of C1orox 20% by volume for 20 minutes~ was the mostefficient treatment used for explant sterilization in the experimentsconducted for exp1ant steri1ization.

The use of Clorox~ 20% by volume for 20 minutes~ was the mostefficient treatment used for explant steri1ization in the experimentsconducted for explant steri1ization.

The use of activated charcoal in the media or added on thesurface of the media inhibited the induction and growth of explants fromdifferent parts of organs of I. grandis~ E. saligna~ E. robusta andE. tereticornis.

Fresh and dry weights of calluses from explants of 1eafbladesof 1eafblades of adu1t~. grandis increased with the increase in thesucrose concentration in the Nash and Oavies (1972) medi um. Root mor-phogenesis occurred in the treatments with 25.0, 30.0, 35.0 and 40.0g/l of sucrose. Most of the roots observed were aeria1.

The occurrence of polarity was observed in the development ofcalluses grown on the Nash and Oavies (1972) medium with high concentra-tions of H3B03. Morphogenesis of aerial roots was observed in thetreatments with 3.2, 6.4, 12.8 and 25.6 mg/l of H3B03.

In the experiments with nitrogen sources (NaN03 and NH4Cl) atvarious concentrations an increase in callus fresh weight was observedfollowing an increase in the concentration of either NaN03 of NH4Cl.Morphogenesis was not observed at the 60th day of culture for bothtreatments in either darkness or in a 12 hour photoperiod.

Exp1ants from leafblades of I. grandis were cultured on the Nashand Oavies (1972) medium with different concentrations of auxins (IAA,IBA, NAA and 2,4-0) and kinetin. Root morphogenesis was observed inseveral treatments: lAA (0.1 and 20.0 mg/l) plus 1.0 mg/l of kinetin;all the treatments of IBA with or without kinetin; NAA (4.0, 8.0 mg/l)alone, (10, 2.0, 4.0 and 8.0 mg/1) plus 0.5 mg/l kinetin, and (2.0, 4.0and 8.0 mg/l) plus 1.0 mg/l kinetin; 2,4-0 (2.0, 4.0 and 8.0 mg/1)a1one, (4.0, and 8.0 mg/1) p1us 1.0 mg/1 kinetin, (1.0,4.0 and 6.0 mg/lplus 4.0 mg/o kinetin and 0.5 mg/l plus 6.0 mg/l kinetin; and kinetin(0.5, 1.0, 2.0, 4.0 and 8.0 mg/l) alone.

The auxin IBA was the most successful in producing root morpho-genesis. Large functional roots were observed early during the second

Callus cultures containing early stages of embryogenesis wereobserved in cultures initiated with 1 year old Eucalyptus grandisexplants grown on the Nash and Davies (1972) medium and then transferredto the Sommer et ~., (1975) medium under a 12 hour photoperiod developedcalluses of different colors (pale, white, yellow, greenish, and red).

Root morphogenesis was observed with or without GA3 pre-treat-ment of the leafblade explants of adult and juvenile Eucalyptus alba,I· camaldulensis, I. robusta, I. saligna and I. grandis grown on theNash and Davies (1972) medium with 1.0 mg/l of IBA substituting the2,4-D and no kinetin. Under either darkness or a 12 hour photoperiod,shoot morphogenesis occurred in culture of I. robusta following the GA3pre-treatment under a 12 hour photoperiod. The GA3 pre:treatment didnot induce root morphogenesis in experiments with the Nash and Davies(1972) medium containing 1.0 mg/l 2,4-D plus 0.5 mg/l kinetin grownunder either darkness or a 12 hour photoperiod.

Coconut milk inhibited root morphogenesis in hypocotyl culturesof Eucalyptus grandis. Cultures containing structure resembling theearly stages of embryogenesis were observed in explantsof leafblade ofadult Eucalyptus grandis cultured on the Nash and Davies (1972) mediumwith 2,4-D, 0.1 mg/l plus 15% of coconut milk under dark conditions.

Cell suspension cultures were established from callus culturesusing a linear shaker and/or roller drum directly from explants. Nomorphogenesis was observed in the cell suspension cultures. However,root morphogenesis was observedin cell suspension cultures of Eucalyptus

grandis. This occurred in seed1ing 1iquid suspension callus cu1turescultured on Sommer et ~., (1975) medium which were subsequently trans-ferred on to Nash and Oavies (1972) solid medium containing 1.0 mg/l2,4-0 and no kinetin. This was observed in the absence of il1umination.The same observation was made in suspension cultures of 1 1/2 year treecal1us cu1tured in Sommer et ~., (1975) No. 1 medium transferred toNash and Oavies (1972) medium with 1.0 mg/l 2,4-0 plus 15% coconut milkand the cultures grown in the dark.

In the experiment using nodal culture, Euca1yptus species had adifferent behavior. Roots and naked buds and/or adventitious budsdeveloped from adult~. robusta, adult and juvenile ~.grandis and adultE. camaldulensis were cultured on Nash and Oavies (1972) medium with IBA.Eucalyptus saligna developed roots only in the experiments with 2,4-0and kinetin and GA3 pre-treatment under il1umination and E. camaldu1ensisdeve10ped naked buds under the same conditions.

Euca1yptus alba did not develop roots or shoots in the noda1culture experiments. However,~. camaldulensis developed shoots in theinternode with GA3 pre-treatment and cultured in the medium with IBAunder a 12 hour photoperiod.

Morphogenesis in Eucalyptus species was reported before by:Aneja and Atal (1969) in cu1ture of lignotuber of ~. citriodora whichp1antlets; de Fossard, (1974) using culture of ~. grandis hypocoty1s,observed root morphogenesis; and Creswel1 and de Fossard (1974), usingnodal culture of I. grandis, observed root development and axillary budgrowth.

Until the presentt no work has been reported with explants ofEucalyptus species leaves or anthers as a source of material for tissueculture techniques. The same is valid for plating of Eucalyptus cellsuspension cultures.

The species specific behavior of Eucalyptus in tissue cultureis the same as that observed in other genera; even in closely relatedspecies such as I. grandis and I. salignat or within the species in thecase of I. grandis with material from seedlingt juvenile and adulttrees.

In the species used in this investigationt primarily ~. grandist

morphogenesis is not specifically controlled by auxin and/or cytokininconcentration ratio in the culture medi um.

Abdul-baki, A.A., 1974: Hypoehlorite and tissue sterilization.Planta. 115:337-6.

Aghion-Prat, D., 1965: Neoformation de fleurs in vitro chez Nicotianatobaeeum L. Physiol. Vegeto 3:229-303.

Amorin, H.V., 1970: lhe effeet of nitrogen and earbohydrate on pro-duetion of phenols by plant eel1 strueture. lhesis, MS, lheOhio State University.

Anagnostakis, S.L., 1974: Haploid plants from anters of tobaeco -enhaneement with ehareoal. Planta. 115:281-3.

Andrede, E.N., 1961: O Euealypto, 2nd Ed. Jundiai: Companhia Paulistade Estrada de Fevro.

Aneja, D and C. Atal, 1969: P1antlet formation in tissue eulture fromlignotubers of Euea1yptus eitriodora, Hook, Current Sei. 38:69-70.

Anonymous, 1948: Vegetative propogation of Euealyptus. Rep. For. limb.Bur. Austr. No. 12.

Aschby, E., 1948: Studies in the morphogenesis of leaves. I. Anessay on leaf shape. New Phytho1. 47:153-176.

Audus, L.J., 1972: P1ant Growth Substanees. Leonard Hi11, London.Baehelard, E.P., 1969: lhe effeets of gibbere11ie aeid on internode

growth and stareh eontents of Euea1yptus eama1du1ensis seed1ings.lhe New Phyto1. 68:1017-22.

and B.B. Stowe, 1963: Rooting of euttings of Aeer rubrum and------_-E.eama1du1ensis Denhn. Austr. J. Bio1. Sei. 16 (4):751-67.

and B.B. Stowe, 1963a: Growth in vitro of roots of Aeer rubrum-------andE. eama1dulensis. Phys. Plant--.16 {1):20-30. -----Bajaj, V.P.S. and M. Bopp, 1971: Gewebeku1tunen in der angewandten

Botanik. Angewandt Bot. 45:115-51.

Baker, R.T. and H.G. Smith, 1920: A research on the eucalypts andtheir essential oils. 2nd Ed. Sydney Gov't. Printer, 470.

Ball, E., 1950: Differentiation in callus cultures of Sequoia semper-virens. Growth, 14:295-325.

______ , 1953: Hydrolysis of sucrose by autoclaving media, a neglectedaspect in technique of cu1ture of peanut tissues. Bul. TorreyBotan, C1ub 80:409-11.

______ , 1955: Studies of the nutrition of ca11us culture of Sequoiasempervirens, An. Biol. 31(3):80-105.

Barnoud, F., 1952: Recherches sur le tissu cambial d'arbres cultivein vitro. These Sc. Universite de Grenob1e.

Bentham, G., 1866: Flora Australiensis. London, Lovell Reeve and Co.VoL 3, viii+70~

Berbee, F., J. Berbee and A. Hi1debrandt, 1972: Induction of ca11usand trees from stem tip cu1tures of hybfid popular. in vitro.7:269.

Bick, I.R.C.; R.B.Brown and W.E. Hil1is, 1972: Three f1avonones from1eaves of Eucalyptus sieberi. Aust. J. Chem. 25:449-51.

Blake, T.J., 1972: Studies on the 1ignotubers of Euealyptus obliqua.L'Herit. 111. The effeets of seasona1 and nutritional faetors ondormant bud development. The New Phytol. 71:327-34.

Blakely, W.E., 1934: ~ ~ to euealypts. 1st Ed. Sydney, The WorkerTrustees.

Boehert, R., 1965: Gibberel1ie acid and rejuvenetion of apiealmeristems in Aeaeia melanoxylon. Naturwissenchaften. 52(3):65-66.

Bode, N.R., 1939: Ueber die Blattausseheidung des Wernuts und ihreWirkung auf andere Plfanzer. Planta. 30:567-89.

Boden, R.W., 1964:. Euealyptus grafting - deve10pment of desirable rootstoeks. Austr. For. Sei. 4(2):173-5.

Bonga, J.M., 1974: Vegetative propogation: tissue and organ eu1ture as ~an a1ternative to rooting euttings. N.Z. J. For. Sei. 4(2):253-60. '

Bottari, F., A. Marsili and I Morelli, 1972: Cyeloartenol, 24-methyleneycloartenol and sistosterol from Euealyptus mierocorys.Phytoehem. 11:2120.

Bove, J.; C. Bove and R. Raveus, 1957: Extration,deparation etdetermination de eertain eomposes hydrosolub1es (glucides solub1es,aeides earboxy1iques non vo1ati1s de C2AC6 et acides amines sol-ub1es) dans 1es p1antu1es et diverses eu1tures de tis sue deCitrus 1imonum. Rev. Gen. Botan. 64:572-92.

Brian, P.W., 1958: Role of gibbere11ie-1ike hormones in regulation ofp1ant growth and f10wering. Nature Lond. 181:1122-1123.

Brown, C.L. and R.H. Laurenee, 1968: Cu1ture of pine ea11us on adefined medium. Forest Sei. 14:62-4.

Brown, S.W., 1944: Studies of deve10pment in 1arkspur. I. Forrosequenee in the first ten mature 1eaves. Bot. Gaz. 106:103-108.

Burgess, 1.0., 1974:. Veget~tive propogation of Euea1yptus grandis.N.Z. J. For. Sel. 4(2).181-4.

Butenko, R.G.; B.P. Strogonov and J.A. Babaeva, 1967: Somaticembryogenesis in earrot tissue eu1ture under eonditions of h;ghsa1t eoncentration in the medium. Dok1. Akad. Nank, SSSR. 1 75:1178-81.

Bychemkova, E., 1964: An investigation of ea11us formation in certa;ntrees and shrubs by the method of tissue eu1ture in vitro. Ooke.Bio1 Seco 141 :1077-80. -

Cam eron, R.J., 1968: The propogation of Pinus radiata by cuttings:inf1uences affecting the rooting of euttings. N.Z. J. For.13:78-89.

Carey, G., 1930: The 1eaf-buds of some woody perennia1s in the NewSouth Wa1es flora Proc. Linn. Soe. N.S.W. 55:708-37.

Carr, S.G.M. and O.J. Carr, 1969: Oi1 glands and ducts in Euca1uptusL'Herit. 1. The ph10em and the pith. Austr. J.Bot. 17:471-513.

Chafe. E. and o. Durzan, 1973: Tannin inc1usions in ce11 suspensioncultures of white spruee. Planta. 113:251-62.

Cha1upa, V., Unpub1ished data.Challenger, S., H.J. Lacey and B.H. Howard, 1965: The demonstration

of root promoting substances in app1e and p1um rootstocks. Ann.Rept. East Mal1ing Res. Sta. for 1964. pp. 124-128.

Chattaway, M.M., 1955: The anatomy of bark. 11. Oi1 glands in Eucalyp-tus species. Austr. J. Bot. 3:21-7.

, 1958: Bud development and 1ignotuber formation in eucalypts.------Austr. J. Bot. 6:103-15.

Cleland, C.F., 1963:cell elongati on.

Hydroxyproline as an inhibitor Qf auxin-inducedNature, 200:908-9.

Claudot, J., 1956: De quelques recherches faits an Marroc concernantles problems foundamentaux. Docum. World. Eucalyptus conf. No.FO/56/Eu-a-1. pp. 4.

Cocking, E.C., 1972: Plant ce11 protoplast isolation and development.Ann. Rev. P1. Physiol. 23:29-50.

Cocoran, M.R., T.H. Geissmann and B.O. Pkiney, 1972: Tannins asgibberellin antagonists. Plant Physiol. 49:323-30.

Cova, V., 1943: Colture di tessuti vegetali IV Ricerche sul contenutodi zuccheri nelli colture de Dancus canota. Atti. Accad. Sci.Torino. 78:203-20.

Cremer, K.W., 1972: Morphology and deve10pment of the primary andaccessory buds of Eucalyptus regnans. Austr. J. Bot. 20:175-95.

Creswell, R.J. and R.A. de Fossard, 1974: Organ culture of Eucalyptusgrandis. Austr. For. 38:55-69.

Cronshaw, F., 1965: Crystal containing bodies of p1ant cells.Photoplasma. 59:319-25.

Crossley, D.I., 1956: Techn. Note For. Br. Cano No. 35 (cited byJ.D. Matthews 1963).

Czsnowski, J., 1952: Badania and gospodarka witaminowa tkanekroslinnych. Hodowanych in vitro na tle ich gospodarki substancjamiwzostowymi typer auksyny--.Poznan. Towarz. Przyjaciol Nank.Wyazial Mat. Przyrod. Prace Kom Biol. 13:209-46.

Dadswell, H.E. and H.D. Ingle, 1951: Wood anatomy in the genusEucalyptopsis White. J. Arnold Arbor, 32:150-1.

Davidson, J., 1974: Reproduction of Eucalyptus deg1upta by cuttings.N.Z. J. For. Sci. 4(2):204-10.

Davie$, D.D., 1961: Intermediary metabo1ism in p1ants. Cambridgemonographs in Exp. Bio1. Cambridge Press. 11:108.

Demetriades, S.D., 1958: Sur 11uti1isation des acides amines, commesource unique d1azote, par 1es tissus vegetauz nounaux cultivesin vitro. Ann. Inst. Phytopatho1. Benaki. 1:121-58.

Digby, J. and F. Skoog, 1966: Cytokinin activation of thiamine bio-synthesis in tobacco ca11us cultures. P1ant Physiol. 41:647-52.

Duraton, H., 1959: Etude de metabo1isme de 11arginine libre dans 1esti.ssus du topinambur cultives in vitro. These Sei. Universitede Paris.

_____ ~, and M. Mai11e, 1961: Etude du metabo1isme de la proline chez1e topinambour. Compt. Rend. 253:963-5.

______ , 1961a: Etude du metabo1isme de 1a pro1ine en ro1ation ouec 1syntheses des pigments chez 1e topinambour Compt. Rend. 243:963-5.

_______, 1962: Metabo1isme de 1a pro1ine chez 1e topinambour. Ann.Physio1. Vegeta1e. 4:271-94.

Durzan, D.S., S, Chafe, and S. Lopushanski, 1973: Effects of environ-mental changes in sugars, tannins, and organized growth in suspen-sion cu1tures of white spruce. Planta. 113:241-9.

_______, and R.A. Campbe11, 1974: Prospects of the introduction oftraits in forest trees by ce11 and tissue cu1ture. N.Z. J. For.Sei. 4(2)261-6.

Dutt, A.K., C. L. Madan and C.K. Ata1. 1971: Vegetative propogationof euca1ypts. Indian Forester. 6:10-11.

E11iott, B.B. and A.C. Leopo1d. 1963: An inhibitor of germinationand of amy1ase activity in oat seeds. P1ant Phys;ol. 6:66-78.

Eng1er, A, and K. Prant1, 1893: Die natur1ichen Pf1anzenfami1ien.Leipzig, Wi1he1m Eng1emann. Tei1 3, V+274.

Esan, E.B., 1973: A detai1ed study of adventive embryogenesis in theRuthacea. Ph.D. Thesis, Univ. Ca1if.

Fazio, S., 1964: Propogation by cuttings. Proc. P1. Prop. Soe. 14:1964-1965. (86.8, 156-62, 280-90).

Fie1ding, J.M., 1948: The breeding of indigenous Australian trees.Austr. For. 1(2):75~81.

Fine, M., 1968: The contro1 of tracheary e1ement formation in Euca1yp-tus tissue cu1tures. Ph.D. Thesis. Vale Univ.

Fosket, D.E. and J.G. Torrey, 1969: Hormona1 contro1 of ce11 pro1ifer-ation and xy1em differentiation in cu1tured tissues of G1ycinemax varo Bio1oxi. Pl. Physio1. Lancaster. 48:871-880.

Fossard, R.A. de, R.J. Creswe11, E.C.M. Lee and R.A. Bourne, 1972:Tissue and organ cu1ture of Euca1yptus species. In Int. Assoe.P1ant Tissue Cu1ture Newsletter. No. 6:10-11.

_______, C. Nitsch, R.J. Creswell, and E.C.M. Lee, 1974: Tissue andorgan culture of Eucalyptus. N.Z. J. For. Sei ..4(2):267-78.

______ , 1974: Tissue culture of Eucalyptus. Austr. For. 38:43-54.Franclet, A., 1963: Improvement of Eucalyptus plantations by vegeta-

tive propogation. FAO World Consult. For. Genet. Stockho1m.No. FAO/FORGEN.

______ o 1970: Techniques of the propogation of Eucalyptus camaldulen-~ by cutting. Note Tech. Inst. Rebois. Tunis No. 12. 55 pp.

Fuchs, Y. and M. Lieberman, 1968: Effects of kinetin, lAA, andgibbere11in on ethylene production and their interations ongrowth of seedlings. Plant Physiol. 43: 2029-36.

Gautheret, R.J., 1934: Culture du tissu cambial. C.R. Herd. Seanc.Acad. Sei. 198:2195-6.

_______, 1935: Recherches sur la culture de tissus vegetaux: essaisan culture de quelques tissus meristematiques. These Sei. Univer-site de Paris.

______ , 1937: Nouvelles recherches sur la cultive du tissu cambial.Compt. Rend. 206:572-4.

______ , 1941: Action du saccharose sur la croissance des tissus decarotte. Compt. Rend. Soe. Biol. 135:875-7.

_______, 1945: Une voie nouvel1e en biologie vegetale: la cultive destissus. Gal1inard, Paris.

______ , 1966: Factors affecting differentiation of plant tissues grownin vitro. In Cell Differentiation and Morphogenesis. W. BeermannTed.) North Holland Pub. Co. pp. 55-71.

Gibbs, R.D., 1974: Chemotaxonomy of flowering plants. McGill-Queen'sUniv. Press. London. Vol. 111, 19aO.

Giordano, E. 1960: Vegetative propogation of Euca1yptus. Docum. 4thSess. Wkg. Party on Eucalyptus, FAO Jt. Subcomm. Medit. For. ProboLisbon, 1960. No. FAO/SCM/EU/60-7c. 6pp.

_______, 1960a: La propagazione vegetative degli eucalitti. Cellu10sae Carta. 11(12):5-10.

_______, 1961: L'impiego della nebullizzazione nel radicamento de11etalee degli eucalitti. Cellulosa e Carta. 12(12):6-9.

Girouard, R.M., 1967: Anatomical and biochemical studies of rootingin stem cuttings of Hedera helix I. Ph.D. Thesis. Purdue Univ.Lafayette. Indiana.

, 1969: Physi10gica1 and biochemica1 studies of adventicous-------rootformation: extracti1e rooting cofactors from Hedera he1ix.

Cano Journ. Bot. 47:687-99._______, and C.E. Hess, 1966: Distribution and moti1ity of rooting

cofactors in juveni1e adu1t growth phases of Hedera helix. Proc.17th Intern. Hort. Congr. Mary1and. Vol. I summary of paper 370.

Goebe1, K., 1898: Organographie der Pf1ansen. 1rst. ed. Jena._______, 1928: organographie der Pf1ansen. 3rd. ed. Jena.Goris, A., 1947: Hydration de fragments de tubercu1es de carotte et

de topinanbom cultives in vitro sen mi1ieux depourvus de sucres;inf1uence de 1 'acide indo1e-3-acetique. Compt. Rend. Soe. Bio1.141:1205-7.

_______, 1948: Epuisement des reserves glucidiques de fragments detubercu1es de topinambom cultives in vitro sur mi1ieux depourvas-de sucres: influence de l'acide indo1e-3-acetique. Compt. Rend.226:742-4.

_______, 1948: Epuisement des reserves glucidiques de fragments detuberc1es de carotte maintenus in vitro surmi1ineux depourvus dessucres. Compt. Rend. 226:105-7.

,1950: Inf1uence componee de l'acide indo1e-acetique et du------~laitde Coco sur 1es reserves glucidiques de tissu de Carotte.Compt. Rend. 231:870-2.

, 1954: Transformations glucidiques intratissu1ares. Anne.------Biol. 30(3):297-318._______, and L. Duhamet, 1958: Etude de l'action du 1ait de Coco sere

1a croisance et la composition glucidi qu des tissus vegetauxcultives in vitro. Rev. Gen. Botan. 65:5-48.

Gram, K. & C.S. Larsen, 1960: The flowering of teak (Tectona ~randis)in aspects of tree breeding, based on observations ;n Thalland.(Docum). 3rd. Sess. FAO Teak Sub-Comm. New Delhi. No. FAC/TSC60/3.2. 1960.

Guha, S. and S.C. Maheshwaii, 1961: Deve10pment of embryoids frompo11en grains of Datura in vitro. Phytomorph. 17:454-61.

Ghugale, D., D. Ku1karni and R. Narasimham, 1971: Effect of auxinsand gibberel1ic acid on growth and differentiation of Morus a1baand Populus nigra tissues in vitro. Indian J. Exp. Biol. 9:381-4.

Gurge1 Filho, o.A., 1959: A propagaçáo vegetativa de espécies flore-stais (1) Rev. Agric. Piracicaba. 34(1):11-30+2 tbls.

Hacket, W., Unpublished data.Halperin, W. and D.F. Wehterell, 1964: Adventive embryogeny in tissue

cultures of the wild carrot, Daucus carota. Am. J. Bot. 51:274-33._______, 1966: Single cells, coconut milk and embryogenesis in vitro

Science. 153:1287-8.Harris, A.D., 1955: Natural regeneration of Farrah forests. Paper

presented to Australian and New Zealand Association for theAdvancement of Science. Melbourne.

Hartman, H.T. and D.E. Kester, 1968: Plant Propogation-Principles andPractices. 2nd Ed. Prentice-Hall, Inc. Eng1ewood, New Jersey.702 pp.

Harvey, A.E. and J.L. Graham, 1969: Procedures and Media for Obtainingtissue cultures of 12 conifer species. Cano Bot. 47:547-49.

Haskins, F.A., and H.J. Gorz, 1961:acids in sweet clover extracts.6:298-303.

Assay of EIS and O-hydroycinnamicBiochem. Biophys. Res. Comm.

Heller, R., 1953: Recherches sen la nutrition minerale des tissusvegetaux cultives in vitro. Compt. Rend. 241:234-6.

Hel1er, R., 1965: Some aspects of the inorganic nutrition of p1anttissue cu1tures. Proc. Int. Conf. P1ant Tissue Culto Penn.State. Univ. 1963. pp. 1-17. McCutchan Pub. CO. Berk1ey.

Henderson, J.H.M., 1954: The changing nutritional pattern from normalto habituated sunflower ca11us tissue in vitro. Annu. Bio1.30(3):329-48.

Hess, C.E., 1957: A physio10gical ana1ysis of rooting in cutting ofjuvenile and mature Hedera helix L., Thesis, Corne11 Univ.Ithaca.

_______, 1959: A study of p1ant growth substances in easy and diffi-cu1t-to-rootcuttings. Proc. Plant Propago Soc. 9:39-45.

_______, 1961: The physio10gy of root initiation in easy-and difficu1t-to-root cuttings. Homo1. 3:3-6. Pub1ished by Arnchem Products Inc.Amb1es. Penn.

______ , 1962a: Physio10gica1 and ana1ysis of root initiation in easy-and difficu1t-to-root cuttings. Rep. 15th Intern. Hort. Congr.Brusse1s. pp. 375-81.

______ , 1962b: Characterization of rooting cofactors extracted fromHedera he1ix L. and Hibiscus rosasinensis L. Proc. P1ant PropagoSoe. for 1961. pp. 51-7. A1so in Rept. 16th Intern. Hort. Congr.Brussels pp. 382-88.

_______, 1962C: A physio10giea1 eomparison of rooting and easy-anddiffieu1t-to-root euttings. Proe. P1ant Propago Soe. pp. 265-268.

_______, 1964: Natural oeeuring substanees whieh simu1ate rootinitiation. Regu1aterms nature1s de 1a eroissanee vegeta1e.Edited by J/P. Nitseh. C.N.R.S. Gif sur-Yveta pp. 517-527.

_______, 1965: Pheno1ie eompounds as stimu1ators of root initiation.P1ant Physio1. Supp1. 40:XLV.

_______, 1966: Root initiation and juveni1ity - a possib1e imp1ieationof terpene eompounds. Proe. 17th Intern. Congr. Mary1and,Vo1. 3pp. 443-451.

Hi1debrandt, A.C., A.J. Riker and B.M. Duggar, 1946: The inf1ueneeofthe.eomposition of the medium in growth in vitro of exeised tobaeeo andsunf10wer tlssue eu1tures. Am. J. Bot:""33:591-7.

_______, and A.J. Riker, 1949: The inf1uenee of various earbon com-pounds on the growth of marigo1d, Paris-daisy, periwink1e, sun-f10wer and tobaeeo tissue in vitro. Am. J. Bot. 36:75-78.

_______, and A.J. Riker, 1953: Inf1uenee of eoneentrations of sugarsand pó1ysaceharides on ca11us tissue growth in vitro. Am. J. Bot.40:66-76. -

_______, 1971: Growth and differentiation of sing1e ee11 and tissues.In IILeseu1tures de tissus de p1antes.1I Edition du eentre Nat.de 1a Rech. Sei. Paris. pp. 71-93.

Hi11is, W.E., 1966a: Variation in po1ypheno1 eomposition withinspeeies of Euea1yptus. L'Herit. Phytoehem. 5:541-56.

_______, 1966b: Po1ypheno1s in the 1eaves of Euea1yptus. L'Herit: Aehemotaxonomie survey - I. Phytoehem. 5: 1075-90.

_______, and N. Ishikura, 1969: An enzyme from Euea1yptus whieh eonvertsto einnamoy1 triaeetie aeid into pinosy1vin. Phytoehem. 8:1079-88.

, and H. Morita, 1969: Po1ypheno1s in the leaves of Euea1yptus.------Renantherin and Macrantherin. Austr. J. Chem. 22:1471-6._______, and Y. Yazaki, 1973: Kinos of Euea1yptus speeies and their

aeid degradation produets. Phytoehem. 13:495-8._______, and Y. Yazaki, 1973a: Properties of some methy1e11agie aeids

and their glyeosides. Phytoehem. 12:2963-8._______, and Y. Yazaki, 1973b: Wood po1yphenols of Euea1yptus polyan~

themos. Phytoehem. 13:2969-77.

Hu, C. and I. Sussex, 1971: In vitro deve10pment of embryoids oncoty1edons of I1ex aguifo1ium. Phytomorph. 21:103-7.

Huhtinen, O., Unpub1ished data.Ing1e, H.D., and H.E. Dadswe11, 1953: The anatomy of the

timbers of the Southwest Pacific area. 111. Myrtaceae. Austr.J. Bot. 1:353-400.

Isikawa, H., S. Yazawa and J. Hanaway, 1963: Some cyto10gica1 obser-vations on a cu1tured ca11us of Pinus strobus. 74th Meet. Jap.For. Soe. 227-9.

_______, 1967: A brief history and recent achievements on the steri1ecu1ture of shoot apices from the practica1 point of view. Jap.For. Soe. J. 49:391-7.

_______, 1972: Stem tip cu1ture and ca11us cu1tures of Crypotomeriajaponica. Third Symp. P1ant Tissue Cu1t. Japan. 5.

, 1973: Invitro information of adventitious buds and roots on-------hypocoty1cu1tured of Crypotomeria japonica. Proc. 38th. Ann.

Meet. Bot. Soe. Japan. 57._______, 1973: (in press). In vitro formation of adventitious buds and

roots on hypocoty1 of Cryptomeriajaponica. Bot. Mag. Tokyo.1105.

Jacobs, G., P. A11an and C.H. Bornrnan, 1970: Tissue cu1ture studieson rose: useof shoot tip exp1ants. 2. Cytokinin: gibbere11ineffects. Agrop1anta1. 2:25-8.

Jacobs, M.R., 1935: lhe primary and secondary1eaf-bearing systems ofthe euca1ypts and their silvicu1tural·significance. Bee1l. For.Bur. Austr. 18:1-78.

_______, 1955: Growth habits oftheeuca1ypt5~ Canaberra Commonwea1thGovt. Printer. 1-262.

Jacquiot, C., 1951: Action du meso-inosito1 et de 1 'adenine sur 1aformation de burgeons par 1e tissu cambial .d'Ulmus campestriscultives in vitro. Compt. Rend. 233:815-17.

_______, 1955: Formation d'organes par 1e tissu cambial d'U1muscampestris. L. et de Betu1a verrucosa gaertn. cu1tivesin vitro

______ , 1959a: Contribution to the study of organogenesis from1iquified p1ant tissue cu1tivated in vitro. 84th Conf. Soe.Savanthes. 441-9. --

_______, 1959b: App1ication of p1ant tissue cu1ture techniques tosevera1 prob1ems of tree physio10gy. Ann. Sei. For. 21:317-473.

, 1964: Structure of excised roots orof organs formed de novo---from cambial tissue of trees grown in culture. Rev. Cyto'f:""Bio1.

Veg. 27:319-22.Kameya, T. and K.H. Hinata, 1970: Induction of hap10id plants from

pol1en grains of Brassica. Jap. J. Breeding. 29:82-7.Kefford, N.D., J.A. Zwar and M.I. Bruce, 1968: Antagonism of purine

and urea cytokinin activities by deviatives of benzylurea. InBiochemistry and Physiology of Plant Growth Substances. (Wightmanand G. Setterfie1d, eds.) Runge Press, Ottawa.

Kitahara, E. and L. Caldas, unpublished data.Kochba, J. and P. Spiegel-Roy, 1972: Effect of cu1ture media on

embryoid formation from ovu1ar ca11us of Sharmouti orange.(Citrus senensis). Zeits. fuer Pflanzenzuedr. 69:156.

___ , J. Button, P. Spiege1-Roy, C.H. Bormann and M. Kochba, 1974:Stimu1ation of rooting Citrus embryoids by gibberel1ic acid andadenine sulphate. Am. Bot. 38:795-802.

Koehler, D. and A. Lang, 1963: Evidence of substances in higher plantsinterfering gibberellin responses. P1ant Physiol. 38:555-60.

Konar, R.N. and Y.P. Oberoi, 1965: In vitro development of embryoidson the cotyledons of Biota orientalis. Phythomorph. 15:137-9.

Kranz, C., 1931: Kenntnis der weechselender blatform des eeus undihrer ursachen. Flora. 25:289-320.

Langner, W., 1964: The origins of juveni1e forms in Chamaecyparis.Silvae Genetica. 12(3):57-63.

Lavee, S., Unpublished data.Lee, E.C.M. and R.A. de Fossard, 1974: The effects of various anxins

and cytokinins on the in vitro culture of stem and lignotubertissues of Eucalyptus bancroftii. Maiden. New Phytol. 73:707-17.

Leopold, A.C. and P.E. Kriedemann, 1975: P1ant Growth and Development.2nd Ed. McGraw-Hill, Inc.

Lethan, D.S., 1963: Regulators of ee11 division in plant tissue.I:N.Z. J. Sei. 1:336-49.

Leshen, Y., 1973: The Molecular and Hormonal Basis of Plant GrowthRegulation. Pergamon Press Ltd. Oxford.

Lingappa, Y., 1957: Tissue culture of Solanum tuberosum and Ipomeapandurata. Am. J. Bot. 44:419-23.

Linsmaier, E.M. and F. Skoog, 1965: Organie growth faetor require-ments of tobacco tissue cultures. Physio1. P1antarum. 18:100-27.

Leuchmann, J.G., 1898: A short dichotomous key to the hiterto knownspecies of Euca1yptus. Rep. Austr. Adv. Sei. 7:523-36.

Maracvi11aca, M.C. and E.R. 'Montaldi, 1963: Rooting of Euealyptusrostrata, FAO World Consulto For. Genet. Stockho1m. No. FAO/FORGEN. 63-5/8. .

_______, and E.R. Monta1di, 1964: The rooting of Euca~yptus rostrata.Idia. Suppl. No. 12:65-72.

, and E.R. Monta1di, 1964a: Cultivo in vitro de tejidos de-------tejidosde euca1ypto. Idia. Supp1. No--.12:62-4.Maiden, J.H., 1889a: The examination of kinos as an aid to the

diagnosis of euca1ypts, Part I. The ruby group. Proc. Linn. Soe.N.S.W. 4:605-18.

_______, 1889b: The examination of kins as an aid to the diagnosis ofeuca1ypts, Part 11. The gummy group. Proc. Linn. Soe. N.S.W. 4:1277-87.

_______, 1891: The examination of kinos as an aid to the diagnosis ofeuca1ypts, Part 111. The turbid group. Proc. Linn. Soe. N.S.W.6:389-426.

_______, 1909-33: A critica1 revision of the genus Euca1yptus.Sydney Govt. Printer. 1:349; 2:312; 3:223; 4:343; 5:332; 6:610;7:492; and 8:354.

Mathes, M., 1964: Use of iso1ated p1ant tissues in studies re1ated toforest geneties. Tappi. 47:710-13.

Matsumoto, T., K. Okumishi, K. Nishida, M. Noguchi and E. Tamaki,1971: Studies on cu1ture conditions of higher p1ant ce11s insuspension cu1ture. 11. Effect of nutritiona1 factors on growth.Agr. Bio1. -Chem. 35:543-51.

McAlpine, o. and J.R. Remfrey, 1890: The transverse sections ofpetio1es of euca1ypts as an aid in the determination of species.Proc. Roy. Soe. Vict. 21:1-64.

McRae, O.H. and J. Bonner, 1953: Chemica1 structure and anti-auxinactivity. Physio1. Planto 6:485-510.

Menoret, Y. and G. More1, 1958: Inf1uence de l'acide 2,4-0ieh1oro-phenoxyacetique sur 1e metabi1sme des acides amines libres detissu de Carotte cultives in vitro. Compt. Rend. 246:2625-5.

Michniewicz, M., 1967: The dynamics of gibberellin-like substancesand growth inhibitors in ontogeny of conifers. Wis. F. Univ.Rostock. 16:577-83.

Milborrow, B.V., 1968: Identification and measurement of abscisicacid in plants. In Biochemistry and Physiology of Plant GrowthSubstances (F.W. Wightman and G. Setterfield, eds.) RungePress, Ottawa. .

Millikan, D.F. and B.N. Ghosh, 1971: Changes in nuc1eic acids withmaturation and senescence in Hedera he1ix. Physio1. P1ant.24:10-13.

Mitra, G. and H. Chaturvedi, 1972: Embryoids and complete plants fromunpo11inated ovaries and from ovu1es of in vitro grown emascu1atedflower buds of Citrus sp. Bu11. Torrey Bot. Club. 99:184-9.

Monaco, L.C., M.R. Sondhal, A. Carvalho, O.J. Crocomo and W.R. Sharp,In Press): App1ication of tissue cu1ture in the improvement ofCoffea.

Moorby, J. and P.F. Wareing, 1963: Ageing in woody p1ants. Ann.Bot. N.S. 27(106):291-309.

Morel, G., Action de 11acide pantothenique sur la croissance destissus d'suberpine cultives in vitro. Compt. Rend. 223:166-8.

Muel1er, C.H., W.H. Mueller and B.L. Haines, 1964: Volatile growthinhibitors produced by aromatic shrubs. Science, 143:471-3.

Mue11er, F. von, 1879-84: Eucalyptographia. Melbourne Govt. Printer.Decades 1-10: 1-489.

Mueller, K.C., 1937: Ueber die geschlechtsvertei1ung und den eintritder geschlechtsreife bei der waldkiefer (Pinus sylvestris L.)Zeitschrift fur Forst and Jagdwessen. 69:177-201.

Murashige, T. and J.B. Jones (In Press): Proc. 3rd. Int. Symp. VirusDiseases Ornamental Plants.

_____ , and F. Skoog, 1962: A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiol. Plantarum.15:473-97.

______ , 1974: Plant propogation through tissue culture. Am Deve. Pl.Physiol. 24:135-66.

Muzik, T.J. and H.J. Cruzado, 1958: Transmission of juvenile rootingability from seedlings to adults of Hevea brasiliensis, Nature.Lond. 181:1288.

Naef, J., 1957: Action de la 1umiere surl 'uti1isation du glucose perles tissus vegetaux cultives in vitro. Compt: Rend. 249:1706-8.

Nair. P.M. and L.C. Vining, 1965: Cinnamic acid hydroxylase in spinach.Phytochem. 4:161-8.

Narayana, R., 1963: Growth and differentiation of Pelargoniumhostorum tissue culture. Ph.D. Tesis, University of Wisconsin,Madison, Wisc.

Nash, D.T. and M.E. Davies, 1972: Some aspects of growth and metabo-1ism of Pau1's Scar1et Rose cel1 suspensions. J. Exp. Bot.23:75-91.

Neish, A.C., 1965: Coumarins, pheny1propanes and lignin. In P1antBiochem. (J. Bonner and J. Varner, eds.). Academic Press. NewYork.

Nicho11s, W., W.D. Crow and D.M. Paton, 1972: Chemistry and physio1ogyof rooting inhibitors in adu1t tissue of Euca1yptus grandis. InProc. 7th Intern. Conf. Plant Growth Substances, Canberra.Edited by O.J. Carro Spring Ver1ag, Ber1in. pp. 324~9.

Nicke11, L.G., and J.G. Torrey, 1969: Crop improvement through p1antce11 and tissue cu1ture. Science. 166:1068-9.

___ , and P.R. Burkho1der, 1950: Atypical growth of p1ants ILGrowth in vitro of cirus tumors of Rumex in relation to tempera-ture, p~and various sources of nitrogen, carbon and sulphur. Am.J. Bot. 37:583-47.

____ , 1952: Vitamin B1 requirement of Rumex virus tumor tissue.Bu11. Torrey Botan, Club. 79:427-30.

____ ,1961: Sur la perte de besions en vitamin B1 par des tissusvegetaux cultives in vitro. Compt. Rend. 253:182-4.

Nitsch, J.P., 1949: Cu1tures of fruits in vitro. Science. 110:499.___ , and C. Nitsch, 1956: Anxin-dependent growth of excised

He1ianthus tuberosus tissues. I. Am. J. Bot. 43:839-51.___ , and C. Nitsch, 1962: Composes phenoliques at croissance

vegeta1e. Ann. Phusiol. Veg. 4:211-25., and C. Nitsch, 1967: The induction of flowering in vitro in

---stem segments of Plumbago indica. L. P1enta. 72:355-70., 1968: Induction in vitro de la floraison chez une plante de

---jours comts: P1umbago indica. L. Ann1s. Sci. Nat. (Bot) 9:1-92.

Ohyama, K., 1970: Tissue eulture in mulberry trees. Japan. Agr. Res.Quart. 5:30-4.

_____ , 1972: Tissue eulture in mulberry trees. Japan. Agr. Res.Quart. 5:30.

Oka, S. and K. Ohyama, 1972: Adventitious bud initiation on theeultured ea11us of Broussenetia kazinoki. 3rd Symp. P1ant TissueCu1ture. Japan.

Okuse, I., 1968: Cu1tivation of app1e mesoearp tissue in vitro.Shokubutsu no Kagaku-Chosetsu. 3:56.

Oshigane, K., 1973: Studies on organ formation from Morus calluscu1tured in vitro. 11. Morus hypocty1 euttings cultured in vitro.43rd Meet. Rep. Jap. Si1vieu1t. Sei. 87.

Overbeek, J. van, R. Blondeau and V. Horne, 1951: Transcinnamic acidas an anti-auxin. Am. J. Bot. 38:589-95.

, M.E. Conk1in, A.F. B1akes1ee, 1942: Cu1tivation in vitro-------ofsma1l Datura embryos. Amer. J. Bot. 29:472-7. -- ----Paranjothy, K., Unpub1ished data.Paton, D.M., R.R. Wil1ing, W. Nicho11s and L.D. Pryor, 1970: Rooting

of stem cuttings of Eucalyptus: a rooting inhibitor in adulttissue. Austr. J. Bot. 18:175-85.

Penfo1d, A.R. and J.L. Wil1is, 1961: The euca1ypts: botany, cu1ti-vation, chemistry, and uti1ization. London. Leonard Hil1 (Books)Ltd. XX+551.

Pierick, R.L.M., 1967: Regeneration, venera1ization and flowering inLunaria annua in vitro. Meded. Lamdb. Hoogesch. Wageningen.67:60-71.

Pike, K.M., 1956: Po11en morphology of Myrtaceae from the South-WestPacific area. Austr. J. Bot. 4:13-53.

Piton, F., 1969: Quelques observations cito1ogiques sur des tissusd'Eucalyptus camaldulensis cultives in vitro. Rev. Gen. Bot.76:287-307.

Poidevic, N., 1965: Inhibition of the germination of mustard seeds byunsaturated fatty acids. Phytochem. 4:525.

Pol1ard, J.K., E.M. Shantz and F.C. Steward, 1961: Hexitols in eoeonutmi1k: their role in nurture of dividing ce11s. Plant Physiol.36:492-501.

Pridham, J.B., 1965: Low mo1ecu1ar weight phenols in higher plants.Ann. Rev. Plant Physiol. 16:13-36.

Pryor, L.D. and J.H. Hil1is, lQ54: A new victorian (and South WalesAustralian) eucalypt. Vict. Nat. Melb. 71:125-9.

______ , 1957: The inheritance of some characters in Eucalyptus. Proc.Linn. Soe. N.S.W. 81:299-305. I11ustrated.

______ , 1957: A practical method for the vegetative propogation ofEucalyptus. Proc. Linn. Soe. N.S.W. 82:199-200. Il1ustrated.

, and R.R. Willing, 1963: The vegetative propogation of------euca1ypts. Austr. For. 27(1):52-62.Rao, A.N., 1969: Tissue culture from bulbil of Dioscorea sansibaren~

siso Cano J. Bot. 47:565-66.Reinert, J., 1959: Ueber die kontrolle der morphogeneses und die

induktion von adventivembryonen an gewebekulturem aus Karotten.Planta. 53:318=33.

_______, and H. Schrandolf, 1959: The physiology of tissue cultures oftumor and normal tissue of Picea glanca. Planta. 53:18-24.

Reinert, J., 1963: Experimental modification of organogenesis inplant tissue cultures. In Plant Tissue and Organ Cultures - aSymposium. Int. Soe. Plant Morphologists. Delhi, 168 pp.

____ ,.1973: Aspects of organization-organogenesis and embryogenesis.In Plant Tissue and Cel1 Culture. H.E. Street (ed.) pp. 338-335.

Ricker, A.J. and A.E. Gutsche, 1948: The growth of sunflower tissuein vitro on synthetic media with various organic and inorganicsources of nitrogen. Am. J. Bot. 35:142-54.

Risser, P.G. and P.R. White, 1964: Nutritional requirements of sprucetumor cells in vitro. Physiol. Plantarum. 17:620-35.

Robbins, W.J., 1957a: Physiological aspects of ageing in p1antAmer. Jour. Bot. 44:289-294.

______ , 1957b: Gibberel1ic acid and the reversal of adult Hedera to ajuvenile state. Amer. Jour. 44:743-746.

______ , 1959: Juvenility in trees and shrubs. Abstr. in Proc. 8thIntern. Bot. Congr. Monaco. vol. 2 pp. 328-329.

______ , 1960: Further observatlons on juvenile and adu1t Hedera. Amer.Jour. Bot. 47:485-491.

and A. Hervey, 1970: Tissue eu1ture of ea11us from seed1ing-------andadu1t stages of Hedera he1ix. Amer. Jour. Bot. 57(4):452-

457.Robinson, L.W. and P.F. Wareing, 1968: Experiments on the juveni1e-

adu1t phase ehange in some woody speeies. New Phyto1. 68(1):67-68.

Rogozinska, J., 1967: Tria eonthine, growth substanees and in vitroeu1ture of G1edistsia triaeanthos. L. Bu11. Aead. Po10~ Sei.Ser. Sei. Bio1. 15:313-17.

_______, 1967: The inf1uenee of growth substanees on the organogenesisof honey 10eust shoots. Aeta. Soe. Bo1. Po1. 37:485-91.

Ruggeri, C., 1960: Root formation in euttings of Euea1yptus eama1du-1ensis Dehnh (rostrata). Doeum. 4th Sess. Wkg. Party on Euea1yp-tus. FAO Lt. Subeomm. Medit. For. Prob1. Lisbon, 1960. No.FAO/SCH/EV/60-7a. 3 pp.

_______, 1963: Further researeh on root formation in Euea1yptuseama1du1ensis Denhn (rostrata) euttings. Pub1. Ant. Sper. Agri.For. Roma. 6:239-50.

, 1966: Antomiea1 observations on euttingof Euea1yptus-------eama1du1ensisDenhn (E. rostrata) propogated by hydropon;e.

Pub1. Cento Sper. AgrT. For. Roma. 8:231-40.Sankh1a, N., D. Sankh1a and V. Chatterji, 1967: Produetion of p1ant-

1ets from ea11us derived from root tip exeised embryos ofEphedra fo1iata. Naturuiss. 54:349.

Sastri, R.L.N., 1963: Morphogenesis in p1ant tissue eu1tures. InPlant Tissue and Organ Cu1ture-A Symposium. Int. Soe. P1ant Morpho-10gists, De1hi. pp. 105-7.

Sato, T., 1974: Ca11us induetion and organ differentiation in anthereu1ture of pop1ars. J. Jap. For. Soe. 56:55-62.

Séhaffa1itzky de Muehade11, M., 1954: Juveni1e stages in woody p1ants.Physio1. P1ant. 7(14):782-796.

_______, 1959: Investigations on aging of apiea1 meristems in woodp1ants and ists importanee in Si1vieu1ture. Forst1ige Forgev.Denmark. 25:319-455.

Sehmidt, W.A.K., and W. Brueker, 1959: Fur ku1tur und histo1ogie desstenge1gewebes von Datura innoxia mi11 in vitro. Flora (Lena)147:133-55.

, and W. Brucker, 1959a: .Fum wachstum des crown-ga11 geuebes------von Oatura innoxia nu11 in vitro. Flora (Lena) 147:242-62.Schroeder, C., 1955: Proliferation of mature fruit pericarp tissue

slices in vitro. Science. 122:601.______ , 1957: Growth of avocado fruit tissue on artificial media.

Calif. Avocado Soe. Yearbook. 1957:165-8._______, 1959: Some aspects of fruit tissue culture as related to

developmental anatomy. Indian J. Hort. 15:267-74._______, 1961: Some morphological aspects of fruit tissue grown in

vitro. Bot. Gaz. 122:198-204.______ , 1963: Induced temperature tolerance of plant tissue in vitro

Nature. 200:1301-2. .-Scurfield, G. and C.W.E. Moore, 1958: Effects of gibberellic acid on

species of Eucalyptus. Nature Lond. 181:1276-1277.Shantz, E.M. and F.C. Steward, 1959:

metabolism of plant cells. VII.cultures under optimal conditions(London) 23:371-90.

Investigations on growth andSources of Nitrogen from tissuefor their growth. Ann. Bot.

Sharp, W.R. and L.S. Caldas, 1972: App1ication of nuclear energy inplant tissue culture. Centro de Energia Nuclear na AgriculturePiracicaba, S.P. Brazil.

Skok, J., 1968: Re1ationship of boron to gibbere11ic acid-inducedpro1iferation in debudded tobacco p1ants. P1ant Physio1. 43:284-8.

Skoog, F., C.Z. Schneider and P. Malan, 1942: Interactions of auxinsin growth and inhibition. Am. J. Bot. 29:568-76.

______ , and C.O. Mi11er, 1957: Chemica1 regulation of growth andorgan formation in plant tissues cultured in vitro. Symp. Soe.Exp. Bio1. 11"118-130. -

_______, and O.J. Armstrong, 1970: Cytokinins. Ann. Rev. P1ant Physio1.21:359-448.

_______, 1971: Aspects of growth factor in interactions in morphogene-sis in tobacco tissue cultures. In Les Cultures de Tissus desPlantes. Co11. Int. du C.N.R.S. No. 193. Paris, pp. 115-136.

Smith, I.K. and L. Fowden, 1966: A study of mimosine toxicity inplants. J. Exp. Bot. 17:750-61.

Smith, R.H. and T. Murashige, 1970: In vitro development of theiso1ated shoot apica1 meristem o~angiosperms. Amer. J. Bot.57:562-8.

Sommer, H.E., 1972: Inf1uence of 2,4-dich1orophenoxyacetic acid onnitrate reductase and protein in wi1d carrot (Daucus carota L.)tissue cu1ture. Ph.D. Thesis. The Ohio State Univ.

_______, C.L. Brown and P.P. Kormanik, 1975: Differentiation ofp1ant1ets in long1eaf pine (Pinus pa1ustris Mi11) tissue cu1turein vitro. Bot. Gaz. 136:196-200.

Staritsky, G., 1970: Embryoid formation in ca11us tissue of coffee.Acta. Bot. Neer1. 19:509-14.

Steinhart, C.E., L.C. Standifer and F. Skoog, 1961: Nutrient require-ment for in vitro growth of spruce tissue. Am. J. Bot. 48:465-72.

_______, L. Anderson and K. Skoog, 1962: Growth promoting effects ofcyc1ito1s on spruce tissue cu1tures. P1ant Physiol. 37:60-66.

Steward, F.C. and S.M. Caplin, 1951: A tissue culture from potatotuber: the synergistic action of 2,4-0 and of coconut milk.Science. 113:518-20.

_______, M.O. Mapes, A.E. Kent and R.D. Holsten, 1964: Growth anddeve10pment of cultured plant cel1s. Science. 143:20-7.

_______, A.E. Kent and M.O. Mapes, 1966: The cu1ture of free plantcel1s and its signification for embryology and morphogenesis.In Current Topics in Developmenta1 Biology. Academic Press. NewYork. pp. 113-54.

, 1969: Plant Qhysiology: ª treatise (F.C. Steward, ed.)-------Volume VB. Academic Press. London.Stiche1, E. 1959: Gleichzeitige induktion von sprossen und wurze1n

an in vitro Kultivierten Gewebestueckchen von Cyc1amen Persicum.Planta. 53:293-317.

Stoutemyer, V.T. and O.K. Britt, 1969: Growth and habituation intissue cu1tures of English ivy, Hedera he1ix. Amer. Jour. Bot.56(2):222-226.

Straus, J. and C.D. LaRue, 1954: Maize endosperm tissue growth invitro. I. cultural requirements. Am. J. Bot. 41:687-94. --

______ , 1962: Invertase in cell wall of plant tissue cu1tures. PlantPhysio1. 37:342-8.

Street, H.E. and G.G. Henshaw, 1966: Introduetion 'and méthods employedin plant tissue eulture. In eells and tissues 1n culture (E.N.Willmer, ed.) Vol. 111. pp. 459-532. Aeademie PreSSa New York.

______ , 1969: Growth in organized and unorganized systems (Chap. 6).In Plant Physiolog. F.A. Steward (ed.). Aeademie Press, New Yorkand London.

Suiter, Filho, W. and J.T. Yonezawa, 1974:saligna grafted by different methods.235-6.

Survival of EuealyptusN.Z. J. for. Sei. 4(2):

______ , 1973: Tissue and Cell Cülture. H.E. Street (ed.) Universityof Calif. Press. Berkley and Los Angeles.

Suda, S., 1960: On the physiologieal properties of mimosine. Bot.Mag. (Tokyo). 73:142-7.

Sussex, I., and M. Clutter, 1959: Seasonal growth periodieity oftissue explants from woody perennial plants in vitro. Seience.129:836-7. --

______ , 1965: The origin and morphogenesis of Eucalyptus eell popula-tion. In Proe. Int. Conf. on Plant Tissue Culture. P.R. White(ed.) McCutchen Pub1ishing Co., Berkley, Ca1if. pp. 383-91.

Syono, K., 1965a: Changes in the response of earrot root callus tothe root extract occurring during their successive cultures,Plant and Ce1l Physiol. 6:285-300., 1965b: Change in organ forming eapacity of earrot root eallus----during subcultures. Pl. Ce1l Physio1. 6:402-19.

Taris, B., 1966: Observations on the behavior of Dothiehiza popu1eaand Cytospora chrysosperma and the deve10pment of each on tissuecultures of Popu1us. C.R. Acad. Sei. 262-0:2347-9.

Tate, R., 1868: A review of the eharacters avai1able for the e1assifi-cation of the euea1ypts with a sinopsis of the speeies arrangedon a capo1ogiea1 basis. Rep. Aust. Asst. Adv. Sei. 7:544-52.

Tay1or, H.f. and R.S. Burden, 1970: Identifieation of p1ant growthinhibitors produced by photo1ysis of vio1axanthin. Phytoehem.9:2217-23.

Tazawa, M. and J. Reinert, 1969: Extra-ee11u1ar and intra-eel1ularehemiea1 environments in re1ation to embryogenesis in vitro.Photop1 asma. 68:157-73. --

Thu1in, J.J. and T. Fau1ds, 1962: Grafting of Euea1yptus, N.Z. J.For. 8(4):664-7.

, and T. Faulds, 1968: The use of cuttings in the breeding and---affore stati on Pinus radiãta N.Z. J. For. ,13:66-77.

Torrey, J.G., 1966: The intiation of organized developmentin plants.Ad. Morphogenesis. 5:39-91.

Trippi, V.S., 1963: Studies on ontogeny and senility in planta l-VIPhyton, Buenos Aires. 20(2):137-174.

Tulecke, W., L. Weinstein, A. Rutner and H. Lamencot, 1962: Biochem-ieal and physiologieal studies of tissue eultures and the plantparts from whieh they were derived. 11. Ginkgo bilboa, L.Contr. Boyce Thom Inst. 21:291-301.

___ , W., R. Taggaret and L. Colavito, 1965: Continuous culture ofhigher plant eel1s in liquid media. Contrib. Boyce ThompsonInst. 23:33-46.

___ , 1967: Studies on tissue cultures derived from Ginkgo bilboa L.Phytomorph. 17:381-6.

Vardjam, M. and J.P. Nitsch, 1961: The regeneration chex Cichorium-endiva L. etude auxines et des kinines endogenes. Bu11. Soe.Bot. Fr. 108:363-374.

Vasil, I.K., 1972: Plants: haploid tissue eu1tures in "Methods andapp1ieation of tissue eulture." P.F. Kruze and M.K. Patterson(eds.). Aeademie Press. New York.

___ , and V. Vsi1, 1972: Totipotency and embriogenesis in plant eel1and tissue eultures. In vitro. 8.

Wareing, P.F., 1958: Reproductive development in Pinus sylvestris.In The Physiology of Forest Trees. ed. K.V. Thimann, pp. 643-54.

____ , 1959: Problems of juveni1ity and flowering in trees. J. Linn.Sce. (Bot) 56(366):282-289.

____ , and L.W. Robinson, 1963: Juvenility prob1ems in wood plants.Rep. For. Res. For. Comm. Lond. 1961/1962. pp. 126-127.

White, C.T., 1951: Some noteworthy Myrtaeeae from the Mo1uccas, NewGuinea and the Solomon IS. J. Arnold. Arbor, 32:139-49.

White, P.R., 1943: A handbook of Plant Tissue Culture. Jaques Catel1Press, Lancaster, Penn.

___ , 1954: The Cu1tivation of Animal and P1ant Ce11s, 1st Ed.Ronald Press. New York.

___ , 1963: The Cultivation of Animal and P1ant Cel1s, 2nd Ed. Rona1dPress, New York:

Winton, L. and D. Einspahr, 1968: The use of heat treated po11en foraspen hap10id produetion. For. Sei. 14:406-7.

, 1972a: Annoted bib1iography of somatie eonnifer ea11us___ o

eultures. Geneties and Physio1ogy Notes No. 16. App1eton,Wise. The Inst. Paper Chem. 19 pp.

_______, 1972b: Bib1iograpnY of somatie ea11us eultures from deeiduoustrees. Geneties and Physiology Notes 'No. 17. App1eton, Wise.The Inst. Paper. Chem. 19 pp.

Winton, L., 1973: Bib1iographie addendum of tree ea11us eu1tures.Genetie and Physiology Notes No. 18. App1eton, Wisc. The Inst.Paper. Chem. 4 pp.

_______, 1974: Seeond addendum to the bib1iography of tree calluscu1tures. Geneties and physio10gy Notes No. 19. App1eton, Wisc.The Inst. Paper Chem. 33 pp.

, Unpub1ished data.-----Wo1ter, K.E., 1964:

callus tissue.In vitro eu1tivation of ash, aspen and pin oakPh.D. Thesis. University of Wiseonsin.

, 1968: Root and shoot initiation in aspen ca11us cu1tures.---Nature. 219:509-10.Wood, H.N., B.A.C. Braun, H. Brandes and H. Kende, 1964: Studies on

the distribution and properties of a new e1ass of eel1 promotingsubstanees from higher p1ant species. Proe. Nat. Acad. Sei. 62:349-56.

Woyeieki, S., 1954: On the origin of the retinospora forms in Thuja,Biota and Chamaeeyparis. Aeta Soe. Bot. Po10n. 23(3):443-458.

Wu, L. and H.W. Zi, 1971: Induction of ea11us tissues. Initiationfrom different somatie organs of riee p1ants by various eoneentra-tions of 2,4-D. Cyto10gia 36:411-16.

Yamada, Y., T. Nishi, T. Yassuda and E. Takahashi, 1967: Steri1ecu1ture of ruee ee11s, Ouxza sativa, and its app1ieation. Adv.Germfree Res. Guotobio1. Proe. Int. Symp. Mijakawa (ed.) CRC Press,C1eve1and. pp. 377-386. '

Yatazawa, M., and K. Furuhashi, 1968: Nitrogen sourees for the growthof riee ea11us tissue. Soi1 Sei. and Plant Nutr. 14:73-9.

Yokoyama, T., 1973: Organ formation in Dios ros Kaki ea11us andeffects of p1ant growth regulators. Chemieal Regu1ation ofPlants). 8:97.

Zenk, M.H., and G. Mue11er, 1964: Biosynthese von 2,6-hydroxybenzoisaureand anderen benzoesaueuren in hoeheren pflanzen. F. Naturforsh.19:398-405.