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Pow .19.} “VI. .1 I. u‘kFl I .45.;oav...‘ Al.143:l.rMOv .Yto .1 o... A 4...]?013t. .5 .TO . I l ,( ...‘.. I 4 ;I ”it... I! '3 v 7', v 3": j: b u. u G l 23 l .m‘ 1%. :luwrw.\ V Hun“. !u.\“uuflllslt|.|lull{l-u\nt 05“ . ’E :Dzi.’ :FE‘I; .3 n'! 0 t" c 1 \11‘ J ¥ .3 _h l"% ‘_.':‘:‘ . r , ‘ ’I ‘ ‘1‘] I, H n 1'?" 0‘: 353’s." av ‘ :'. "if? » . ‘ ' I’L; {Va 2‘ ,1 .2: .‘ :"::'~ v.2: :1: Mg“; ~ " ‘l“ 'o ‘3 ‘ " .. .ntn'nhatccuuu. . . H 1 Hi‘. > i’vh‘l; ' W? L l i t II n .10 1011...: ‘11., M.“ t i:a.t..u‘t‘lu€..i.vt.i:: |!.n1;,I.il . 3%}.ofll‘vfilifihtuf N4$vu§6fllffl .nfiflti flu. \. trill 0.315..) \91? 3.41. l.¢l.houuK¢W.l\.t.-4.$4.kufi3~0 ll ' n 1 ‘ 1:. [it at...~.1z:..‘0 _ fair- ‘ T ”15. .- : v . 4 1:, . ' . .J. ‘.1l..... 3‘.-. _ . . 1.3.6.” '4. .21 .fil..1.|....w.unv.l..- 54,94»).th IIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIZIIIIIIIIIIIIIIIILIII IIIIIIIIIIIIIIIIIIIII Michigan State University This is to certify that the dissertation entitled PROPAGATION OF ACER RUBRUM L. 'RED SUNSET' lfl_VITRO presented by Kent James Nel sh has been accepted towards fulfillment of the requirements for DOCTOR OF PHILOSOPHY _l-|_ORTICULTURE degree in {(fl Major professor ucn'. Alr .‘ 4 - r1 .nrr . , . . 042771 )V1£SI_J RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from _;-—. your record. FINES will be charged if book is returned after the date stamped below. PROPAGATION or gen hymn L. 'RED SUNSET' 111 111139 By Kent James Welsh A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1985 ‘IDLK Ezoo ABSTRACT PROPAGATION OP.A§§3,.3nanu L. 'RED SUNSET' lg VITRO By Kent James Welsh A protocol as developed for propagating Acer rubrum L. 'Red Sunset' innyitzg. A preconditioning treatment which consisted of subculturing shoot tip or node segment explants, 3-” cm, at 3-u day intervals on LS basal medium or with 1.0 mgl"1 BA or Zip was required to establish aseptic, actively growing cultures. The most effective cytokinin for stimulating axillary shoot proliferation was 6-benzylaminopurine (BA) with 5.9 shoots/explant occurring at 5.0 mgl'I. Shoot proliferation was most consistent following decapitation. Incorporation Of 100 mg].'1 adenine sulfate in LS + 10.0 mgl‘1 21p multiplication medium also stimulated axillary shoot proliferation. Rapid growth in yitgg led to the development of vitreous shoots. Vitrification was effectively controlled in 'Red Sunset' shoot tip cultures by harvesting shoots before damage occurred and by culturing explants in 60 m1 bottles rather than 25 x 150 mm culture tubes. 'Red Sunset' shoot tips rooted readily on L8 basal median or on L8 4- IBA, NM or IAA during a 1n day culture period and were easily acclimated to greenhouse conditions. Plants recovered from culture were uniform phenotypically although their leaves were morphologically different from mature, flowering plants. To Laura Lee, Jennifer, Melanie and Erica 1'1 ACKNOWLEDGEMENTS I would like to thank Dr. Kenneth C. Sink for his assistance in this research program and in editing this dissertation. I would also like to thank Dr. Harold Davidson for his assistance with this project. Thanks are also given to Dr. Stanley 0. Howell, Dr. Lowell C. Ewart, Dr. Harry H. Murakishi and Dr. James W. Hanover for their help in the preparation of this dissertation. Thanks and appreciation also go to my wife for her support and patience while the research was being conducted and during the time spent preparing this dissertation. Thanks also go to my parents for the upbringing and encouragement the have given me. TABLE OF CONTENTS Page LIST OF TABLES O O O O O O O O O I O O O O I O O O I O O O O O 0 v LIST OF FIGURfi O O O O O O O O O O O O O O O O O O O O O O O 0 Vi INTRODU CT ION I O O O O O O O O O O O O O O O O O O O O O O O O O 1 LITERAmRE REVIm O I O O O O O 0 O O O O O O I O O O O 0 0 O O 3 Genotype Effects 0 o o o ’0 o o o o o o o o o o o o o o o e 12 Propagation Stages . . . . . . . . . . . . . . . . . . . . 14 swam O O I O O O O O I I O O O I O O 0 O O O O O O O O 26 MATERIALS AND WTHODS O O O O O O O O O O O O O O O O O O O O O 28 cul ture Initiation O O O O O O O O O O O O O O O O O O O O 28 Shoot Proliferation . . . . . . . . . . . . . . . . . . . 29 Rooting, Acclimation and Evaluation . . . . . . . . . . . 30 RESULTS 0 I O I O O O O O O O O O O O O O O O O O O O O O O O O 33 Shoot Proliferation . . . . . . . . . . . . . . . . . . . 36 Shoot Proliferation and Vitrification . . . . . . . . . . H6 Rooting and Plant Evaluation . . . . . . . . . . . . . . . 56 DlstSION O O O O O O O O O O O O O O I O O O O O O O O O O O 0 6n SUM! I O O O O O O O O O O O O O O O O O O O O O O O O O O O 77 BBLImRAmY O O O O O O O O O O O O O O O O O O O I O O O O O I 80 iv LIST OF TABLES Table 1. Woody plant species which have been propagated in m 0 O I O I O O O O O O O O O O O O O O O I O 0 Table 2. The morphogenetic responses of Age: nubzgn_'Red Sunset' Table Table Table Table Table Table shoot tips after 6 wks culture in LS mediun containing the cytokinins, 21p, BA, and kinetin . . 3. The morphogenetic responses of Age: zgpggm 'Red Sunset' shoot tips to decapitation. n. The morphogenetic responses of Age: zupgyn 'Red Suns shoot tips after a 6 wk culture period in LS + 10.0 mgl' The shoot tips were cultured for n wks on LS + 10.0 mgl" 21p, decapitated and basal sections were returned to the same medium with shoots harvested at 10 day intervals . . . . . ‘i‘t' 21p and adenine sulfate or casein hydrolysate . . . . . . . 5. The morphogenetic responses of Agez.znngm_'fied Suns shoot tips after a 6 wk culture period in LS + 10.0 mgl' 21p and varying sucrose or agar concentrations . ?t' 6. The morphogenetic responses of Age: zghzgn_'fled Sunset' or temperature shoot tips to variable salt concentration Basal medium was LS + 10.0 mgl' in the temperature experiment were held for 0-” weeks at Data were taken after a regimes. 20° c then transferred to 25° c. 6 wk culture period . . . . . . . . . 21p. Shoot tips 7. The morphogenetic responses of Age: ngzgm 'Red Sunset' Shoot tips were shoot tips in different 0 cultured in LS + 10.0 mgl‘ Zip. ture vessels. In the six wk experiment, shoot tips were cultured intact for four wks, decapitated two nodes above the base and the basal mass was returned to the same size culture vessel for two weeks . 8. The effect of the auxins, IBA, NAA or IAA incorporated in LS median on rooting of A93: w 'Red Sunset' shoot tips after a 2 wk culture period . . . 37 “3 R7 “9 52 55 57 LIST OF FIGURES Figure 1. Phenolic secretion from Acer rubrum 'Red Sunset' node semnta O O O O O 0 O O O O O 0 O O 0 O O O O O O O O O O 0 Figure 2. Acer rubrum 'Red Sunset' shoot tips. A. Axillary bud break after 11 wks on LS + 10.0 mgl'1 21pi B. Axillary shoot elongation after 6 wks on LS + 10.0 mgl’ 21p . . . . . . . . Figure 3. Growth of Age z.¥ub_gg 'Red Sunset' shoot tips after h wks on LS + 10. 0 mgl' 21p (left) or LS basal medium (right) Figure h. Acer rubrum 'Red Sunset' shoot tip explants 2 wks after decapitation, n. O shoots/explant. The shoot tips were cultured for u wks on LS + 10. 0 mg1'121p, decapitated and returnedtothesamemediunforZwks............ Figure 5. The effect of adenine sulfate on basal callus formation in A992 zghzyn 'Red Sunset' shoot tip cultures after 6 wks on LS + 10. 0 mgl'121p. Significant experiment by adenine sulfate concentration interactions for mean callus fresh weight were observed between the three experiments: 1--,2--0and3---..................... Figure 6. The effect of varying agar concentrations on basal callus formation in M w 'Red Sunset' shoot tip cultures after 6 wks on LS + 10. O mgl"1 21p. A highly significant experiment by agar concentration interaction for mean callus fresh weight was observed between experiment: 1 (0) and 2 (I) . . . . . . . . . . . . . . . . Figure 7. The effect of varying temperature regimes on axillary shoot proliferation in cultures of Age; rubrum 'Red Sunset'. Shoot tip explants were held for o-u wks at 20° C followed by transfer to 25° C for a total of 6 wks. Basal median was LS + 10.0 mgl'1 21p. A significant experiment by temperature interaction in mean shoot nunber was observed between experiment: 1 (0) and 2 (I) . . . . . . . . . . . . Figure 8. Rooted Age; rubrum 'Red Sunset' shoot tips 2 wks after being placed on LS basal medium . . . . . . . . . . . Figure 9. .Aggn.ngbggm 'Red Sunset' plants propagated in vitro; fl wks after transfer from culture to planting medium, height ”.1 m C O O O O O I O I O O O O O O O I O O O O O O O O O 0 vi 3” 38 “0 an ”8 51 5h 58 6O Figure 10. Growth rate of in yitzg propagated Age: rubrum 'Red Sunset' plants in the greenhouse. Four wks after transfer from culture uniform plants were transplanted in 3.8 liter plastic pots using a peat-lite planting mix (VSP, Bay Houston Towing Co.). The plants were fertilized once/week with 150 ppm N, 20-20-20 during the growing period from ”/13/82-8/23/82 . . . . . . . . . . . . . . . . . . . . . . . 62 vii INTRODUCTION Grafting has been used for centuries to propagate selected plant types; many of which are difficult or impossible to propagate by other classical asexual or sexual methods. Physiological disorders sometimes encountered with grafted plants, however, prompt the testing of new propagation techniques. Red Maple, Age; rubrum L., is a prime example of this situation. As a species, A, ggbggm_grows over an extended ecological range covering much of the eastern United States and southeastern Canada (55, 106, 107). Cultivars have been selected from this diverse population for fall coloration, habit, and hardiness. These cultivated types make attractive shade trees and have found use in street plantings, parks and in the home landscape (55). Until recently these cultivars were strictly propagated by budding; with red maple seedlings as the understock (81). Budding red maple is a relatively simple procedure but graft incompatibility often develops. Losses of 501 the first year after budding and an additional 10-201 the second year have been reported (85). The cause of this incompatibility is not known but the genetic diversity of the stock and scion is quite probably involved (20, 55). Propagating these cultivars by cuttage is one obvious way to bypass the incompatibility problem. And, there are a number of reports (17, 20, 30, 31, 58, 81, 85, 97, 113) which describe methods for rooting node or stem tip cuttings of red maple. Propagation by cuttings, however, is 1 2 limited by: 1) the size of the stock block required, 2) seasonal propagation period and 3) the extended period of time required to increase new selections. Micropropagation techniques may be useful in overcoming these limitations (57, 69, 100). To determine the feasibility of propagating red maple in yitgg four objectives were established in this research study: 1) to develop procedures for initiating aseptic cultures using shoot tips from mature, flowering trees, 2) to develop an in yiggg shoot proliferation system, 3) to determine procedures for in yitzg rooting and u) to evaluate the uniformity of plants propagated in yitro. LITERATURE REV IEW In the 80 years since Haberlandt published his work on the culture of isolated plant cells, tissue culture has grown from an enlightened idea to a scientific reality. The isolation and purification of indoleacetic acid (IAA) by Kogl and associates in 193A (10“, 116) and the discovery of 6-furfurylaminopurine (kinetin) (71,72) played a large role in the emergence of this field. The real impetus for tissue culture, however, came with the realization that it was possible to control growth and organization in yitgg by manipulating the ratio of auxin(s) and cytckinin(s) in the culture media (9h). The pioneering work of Skoog and Miller (9h) led to the development of many tissue culture techniques including plant propagation. Woody species have generally been considered to be more difficult to propagate in yitgg than herbaceous species (100), however, evidence is accumulating which refutes this supposition. Abbott (1, 2) suggested that this inconsistency was due to a lack of effort and inadequate research in this area. The rapid increase in the nunber of references dealing with in 11129 propagation of woody species supports this arguement (Table 1). Winton (119) stated that commercial propagation of woody species using tissue culture techniques was hampered by low frequency shoot proliferation and by the use of seedling explants. While this is still true to a certain extent, some success in developing commercially feasible micropropagation protocols has been made in the Eggplaggag 3 n (69)..E£1£§9§§§ (63, 100) and BQSEQQQQ (22,99). And, the number of articles dealing with in 11329 propagation of mature plants and cultivars, including ornamentals has dramatically increased in the last five years. Developing a micropropagation protocol is a matter of trial and error (1) and is dependent on genotype/environment interactions. While there are some general guidelines to follow such as using cytokinin(s) or cytokinin/auxin combinations to stimulate shoot development and auxin(s) to enhance rooting it is not always clear how a particular species will respond. In culture it is possible to closely control and monitor the environment but the genotype or genetic makeup of an individual normally cannot be predictably altered. Therefore, it is important to understand how the genotype responds in a particular environment and how to manipulate the environment to obtain the optimun response. 5 Table 1. Woody plant species which have been propagated ig_vitro. Species Explant Explant Source Reference AW A993: We 'Crimson Sentry' Shoot tips Greenhouse grown 22 trees A. mam seedlings Shoot tips 2 yr-old seedlings 115 'Red Sunset' Nursery grown trees Datum Alnus glutiggsg Lateral buds Seedlings 35 Betgla.pgngglg Stem inter— Seedlings H5 nodes Catkins Mature trees 98 B... We ‘ .gzgghganigg Shoot tips Seedlings 69 Node segments fighygnznggsg Shoot tips 2-20 yr-old trees 19 Stem inter- nodes Leaves .Enisassaa ,Ealniawlatiflglia Shoot tips 3 yr-old seedlings 63 Wu an. Stem segments ‘37 #7ou—792-1u50 Shoot tips 100 Exbury Azalea Shoot tips 33 DH23, DH28 .Iaasinium.ash§i seedlings Shoot tips Seedlings 65,66 'Becky Blue' 'Bluebelle' Mature plants Table 1 (cont'd) Species Explant Explant Source Reference L W Node segments Mature plants 25 .Hnnflgfiflfi Moms alha ygnagiha Leaves Seedlings 80 W Wiles .glahna 'Magnifica' Shoot apices Mature plants 21 Shoot apices Field grown plants 86 .EEQLQEQQBQ .Qnsxillsaian- 'Robyn Gordon' Axillary buds Greenhouse grown 36 plants 'Crosbie Mbrrison' Node segments Field grown plants §_,_ W Node segments 1 yr-old seedlings 10 licensees .Ghaanmles Japgnigg, Shoot tips Field grown plants 79 W 51mm 'Mboncreeper' Shoot tips Field grown plants 79 .Snataszus .pnggnygggntna, Shoot tips Field grown plants 79 .9... x W 'Toba' Shoot tips Field grown plants 79 .Mhlns Rootstocks Antonovka KA313 Stem segments Greenhouse grown 22 plants Table 1 (cont'd) Species Explant Explant Source Reference EMLA 7 EMLA 9 EMLA 27 MAC 9 M 7 Shoot tips #7 M26 - Shoot tips R9 Cultivars 'Stark Jumbo' Stem segments Greenhouse grown 22 plants 'Granny Smith' Greenhouse grown 93 plants 'Jonathan' 'Delicious' .Ma.d9m9§&iga ' 'Golden Delicious Shoot tips Field grown trees 6h immune 'Cox's Orange Pippin' Shoot tips Seedlings 3 Greenhouse grown plants 'McIntosh Meristems Seedlings 110 .fla.§i§hgldiiuznmi 'Calocarpa' Shoot tips Greenhouse grown 87 plants .fl;.§p. 'Almey' Shoot tips Greenhouse grown 87,88 plants 'Dainty' Shoot tips Field grown plants 79 'Golden Hornet' 'Hopa' Shoot tips Greenhouse grown 87 plants .Mo x .RBERNEQE 'Eleyi' Shoot tips Greenhouse grown 87 plants Table 1 (cont'd) Species. Explant Explant Source Reference .Pctsntilla .fnuticcsa 'Coronation Triumph' Shoot tips Field grown plants 79 'Sutter's Gold' .Pznnus Rootstocks Cherry .2£BBH§.§!1HI.X mummy: 'Colt' Stem segments Greenhouse grown 22 plants jghayiyn,F 12/1 Shoot tips 1 yr-old greenhouse #8 grown plants .Eiimahalsh x Mezzard 1h '13, align) Stem segments Greenhouse grown 22 plants Peach .2L.R§£§193 'Nemaguard' Shoot tips 73 Plum .EL.92£B:1£§£a 'Myrobalan' Shoot tips 1 yr-old seedlings 38 lupinsititia 'Pixy' Shoot tips Greenhouse grown 22 plants 1 yr-old plants RB 'St. Julien 1' Stem segments Greenhouse grown 22 plants Table 1 (cont'd) Species Explant Explant Source Reference Cultivars and Species jg_§nyggglg§ Dormant buds Seedlings 70,103 Lmadalus 1: .pggsigg Dormant buds 103 .Bl.azium 'Bing' Dormant buds Field grown plants 96 'Black Tartarian 'Royal Ann' 88am! IL.£§£§§1£2£3 'Atropurpurea' Shoot tips 34 'Newport' Stem segments Greenhouse grown 22 ' plants 'Thundercloud' Shoot tips Field grown plants 79 .2‘.gist§zna Shoot apices Field grown plants 59 hm 'Calita' Shoot tips 83 jg_tgnentg§§ Shoot tips Field grown plants 79 Elnasaniha .cacsinsa 'Lalandei Shoot tips Field grown plants 79 21mm Rootstocks Old Home x Farmingdale 51 Stem segments Greenhouse grown 22 plants Table 1 (cont'd) 10 Species Explant Explant Source Reference Cultivars .El.99nmnn1§ 'Bartlett' Shoot apices Field grown plants 60 'Seckel' Shoot tips Greenhouse grown 88 plants ‘Bgsa.canina Shoot tips Greenhouse grown SH Node segments plants Lateral buds _§; damascena Shoot tips Greenhouse grown 5h Node segments plants Lateral buds .3; hybrida 'Forever Yours' Shoot tips Greenhouse grown 92 plants 'Fragrant Cloud' Buds Greenhouse or field 26 'Garnet Yellow' grown plants 'Improved Blaze' Shoot tips Aseptic stock u1,u6 cultures 'Kings Ransom' Buds Greenhouse or field 26 'L111 Marlene' grown plants 'Parade' 'Pauls Lemon Parade' 'Plentiful' .Bi.h1hnida 'Bridal Pink' Shoot tips Greenhouse grown 5n 'Tropicana' Node segments plants Lateral buds 5.1211193 x L__Inalda 'Anthony Waterer' Shoot apices Field grown plants 59 'Frobelii' Shoot tips Field grown plants 79 Table 1 (cont'd) 11 Species Explant Explant Source Reference .Salicaceae Melba Terminal and 23 axillary buds Shoot tips £5.3lha,x W Terminal and 23 axillary buds Shoot tips L 31113 x m Terminal and 23 axillary buds Shoot tips .2; sausages: Cambium 18 Shoot tips .Ea.§ncnan§£isana Cambium 18 Shoot tips .2..nisza.txniaa, Cambium 13 Shoot tips IL.i£enula Cambium 18 Shoot tips Terminal and 23 axillary buds Shoot tips j;_jm§mnlgig§a Stem segments 118 Terminal and axillary buds Shoot tips Saxifzaaassas .Rib§§_1ngnnigng Shoot tips Greenhouse grown 120 plants 12 GQBQLIEQ.E££29£§ Differential responses between individuals in 11529 can partially be attributed to their genotypes (54). This statement may refer broadly to interfamily differences or to distantly related members of the same family and may also refer to varietal, cultivar, or individual plant differences. These different morphogenetic responses may indicate different growth regulator requirements or a difference in morphogenetic potential. Certain groups of individuals seem to respond more readily in culture than others. This is true of the Bogaggag; Table 1. includes 2h references and covers 28 figsgggggs species. The large amount of research which has been done with this family is due to the horticultural and ornamental value of its members and to the ease with which they can be manipulated in culture. Members of the Rggggggg are generally more responsive to 6-benzylaminopurine (BA) or BA/auxin combinations than to other cytokinins or cytokinin/auxin combinations. The Enigaggag is another family which responds readily in culture. Members of this family which can be propagated in 11.11.29. are: azaleas, Bhodgdendcgns, mountain laurel and blueberries. Shoot proliferation is stimulated in these species by relatively high levels (5-15 mgl'1) of 6(X-X-dimethylallylamino) purine (21p) (6, 25, 33. 57, 63, 65, 66, 100). In comparison there are few published reports of micropropagation of Age: species. This may indicate that members of this family are more ~difficult to manipulate in yitzg than other species. Cheng (22) placed A. platanoides 'Crimson Sentry' with a group of figsgggggs species which were less responsive than others being examined. Welsh et al. (115) reported that it was possible to stimulate axillary shoot development on 13 shoot tips of L rubrum seedlings and 'Red Sunset', however, the results were inconsistent. Differential responses have also been observed between members of the same family. Norton and Boe (79) compared the morphogenetic potentials of 8 flggaggggs species and observed a range of responses from £1.35 'Dainty' with ll shoots/explant to m 'Frobelii' with 38 shoots/explant. The optimun BA concentrations for these 8 species varied from 0.1 to 2.5 mgl". In a separate study (59)..§21£§a.hymalda out yielded Prunus,gist§nna_by more than 10:1 shoots/explant. Closely related species also vary in their morphogenetic responses. Differences in optimum growth regulator requirement and shoot number were observed between Prunus tomentog and §_,_ cerasigega with 2.5 mgl‘1 BA and 15 3.1.u shoots/explant or 0.1 mgl"1 BA and 6 1 0.8 shoots/explant respectively (79). Shoot number also varied between .Qrataegns bragnzagantha with 10 1 2.2 shoots/explant and the cv 'Toba' with 5 ¢_1.u shoots/explant, however, the growth regulator requirement was the same. In a comparison of u crabapple cultivars, Singha (87) observed that the responses of fihlns an. 'Almey'; and 'Hopa' were similar with 7.3 or 7.1 shoots/explant on 1.0 mgl'1 BA.while ML siebgldii_var. zuni 'Calocarpa' and M. x punpnzea 'Eleyi' responded more readily to 2.0 mgl"1 BA with 5.9 or 5.7 shoots/explant, respectively. Gorst et a1. (36) observed variation in nutritional and hormonal requirements and mode of proliferation between two fizeyillga hybrids. Inter and intra specific differences in the nmber of shoots/explant have been observed between Rosa caning. L W, L hybrids 'tropicana' and 'Bridal Pink' (54). Similar differences have been reported with other Rosa cultivars (26, R1). 1n Intra specific differences have also been reported between u blackberry cultivars (111) . These reports make it clear that the response of a plant selection to in yitng conditions is strongly influenced by its genotype. Similar observations have been made with §glan§9ggg§ species (89, 11h). Murashige (75) described 3 stages for in yitng propagation of plants: Stage 1: Culture inititation; Stage 2: Multiplication and Stage 3: Rooting and transfer of plants from culture. While these stages have been recently reviewed (7, nu), they will also be considered here because it is important to understand what occurs in each stage and how they are interbrelated. The introduction of woody plant material to the in yitng culture environment is often complicated by surface or systemic contaminants (36, 67, 7h, 91) and phenolic secretion from excised tissues (1h, 67, 7A, 115). Both of these problems must be overcome to establish aseptic, actively growing cultures. Some tissues are more difficult to surface sterilize than others (91). Fungi and bacteria become trapped in crevices in the bark of explants or at leaf axils and are difficult to dislodge (7h). Pubescent shoots are especially difficult to sterilize because the hairs trap contaminants and prevent penetration of the surface sterilants (91). The use of explants from field grown woody plants represents a significant problem in establishing aseptic cultures (36, 7h, 91). Gorst et a1. (36) initiated cultures of two Qggyillga hybrids and found that field grown material was more difficult to surface sterilize than 15 greenhouse grown plants. They suggested that stock plants should be kept under glass. Strode et al. (100) had to remove Rhododendzgns_frcm humid field conditions and place them in a dry, air conditioned room to obtain clean explants. Earlier, Abbott and Whiteley (3) found that it was not necessary to surface sterilize apical primordia excised from greenhouse grown seedlings and adult flowering trees of Helge sylyestzis 'Cox's Orange Pippin'. Certainly, where possible, the use of greenhouse grown stock plants holds an advantage over field grown plants for culture initation. However, in many instances it may be necessary to use field grown material. Under these conditions, the plant material and/or the surface sterilization procedures must be manipulated to insure establishment of aseptic cultures. The time of year also has an effect on the level of surface contaminants. Skirvin (91) found that peach cuttings taken in the spring while dormant and forced in a warm room were more easily sterilized than those collected later in the year. Others (60, 6h, 80) have used the same procedure to obtain clean explants. A variety of treatments are sometimes used prior to surface sterilization to reduce levels of surface contaminants. Jones and Hopgood ("8) placed Prunus shoot tips under running water for 50 minutes. This procedure reduced the level of contaminants and made them more accessible to sterilizing agents (uh). Dilute detergent solutions, which act as wetting agents, are also commonly used as pretreatments (6, 21, 33, 109). This treatment is especially helpful with pubescent tissues (an). An ethanol dip, normally 70%, may also be employed as a pretreatment (1H, 21, 23, 63). Ethanol acts as a wetting agent (7, 11). Broome and Zimmerman (1”) used a 70% ethanol plus 11 Tween 20, 15 second dip before placing blackberry explants in a bleach solution. Chaturvedi 16 et al. (21) used a sequential pretreatment, with a soak in 5% detergent followed by a quick dip in 951 ethanol. Soaks in fungicides are also used (1"). The pretreatments may be used singly or sequentially depending on the nature of the tissue and the level of surface contamination. Aqueous diluted, commercial bleach, 5.25% sodium hypochlorite, is commonly used as a surface sterilant. It acts by the release of chlorine (11). Other chemicals used as surface sterilants include: calciun hypochlorite (33, 36), 0.01% mercury chloride (19, 21), 70$ ethanol (#5) and 80% isopropancl (96). The concentration and length of treatment varies, depending upon the species, type of tissue, season of year and explant source. In some cases, solutions are agitated (7, 33) or placed under vacuum (7, 33. 91) to encourage better penetration of the surface sterilants. Antibiotics and fungicides have sometimes been included in culture media to eliminate surface and systemic contaminants, but with limited effectiveness and they are sometimes phytotoxic (15). Secretion of polyphenols from out surfaces or damaged tissues is another serious problem encountered in initiating cultures of many woody species. Phenols are released from explants as a result of wounding or injury caused by surface sterilization agents(1u). The brown coloration of damaged tissues and surrounding media is due to the oxidation of polyphenols (113, 67). McComb (67) suggested that phenols could be controlled by adding polyvimrlpyrilodone (PVP) to the culture mediun or by using antioxidants. Welsh et al. (115) however, reported that PVP, ascorbic acid and L-cysteine were not effective in controlling phenolic secretion from Acer rubrum shoot tips. Broome and Zimmerman (1”) reported that 17 soaking blackberry explants in ascorbic acid, citric acid or cysteine HC1 following surface sterilization resulted in further injury. Control of phenolic secretion has been reported by transferring tissues to fresh media every one or two days (1n, 67). Cheng (22) used a "preconditioning treatment" to control phenolic secretion. The treatment consisted of culturing explants on basal medium for one week followed by dissection into 3-10 mm segments and transfer to appropriate media. This treatment was used to eliminate contaminated or injured tissues and to select actively growing explants. This procedure was also found to be effective by Welsh et al. (115). Culturing primary explants of blackberry (111) or blueberry (65) in liquid media on a rotating wheel has also been shown to be effective in controlling blackening of explants. Induction and maintenance of rapid shoot multiplication are the main objectives of Stage 2 (75). In some instances this may occur during the initial culture period although it may take up to two years to acclimate some woody species (68). Abbott and Whiteley (3) reported that shoot tips from mature 'Cox's Orange Pippin' trees required eight weeks culture before shoot proliferation began. Subculture of these in 11329 induced shoots led to a higher proliferation rate. Similar increases in shoot proliferation have been observed with Rubus (1A), .Prnnns (A8), fistula (69) and Rosa (“0) cultures. Hasegawa (A1) suggested that the increased responsiveness of Rosa hybrida shoot tips was due to a change in the physiological state of the explants. Murashige (77) and Winton (119) suggested that it may be necessary to return explants to a juvenile state before regeneration of woody species can be achieved. Juvenile tissues are generally thought to have a higher morphogenetic potential in culture than tissues from mature 18 plants (1h, 67, 99). This may be partially due to the physical condition of the explants. Stokes (99) observed that it was easier to initiate cultures of woody species during early stages of active growth, before the tissues had begun to lignify. Thus, returning mature explants to an active growth phase may stimulate shoot proliferation. Rejuvenation has been reported in cultures of fistula (#5), M313: (93), [applying (65, 66) and Iitla (90). Symptoms of rejuvenation include: changes in morphological characters (65, 90, 93), increased growth and shoot proliferation rates (65, 66) and improved root formation (65, 66 93). Skene and Barlass (90) observed that 1131: shoots grown from mature 'Cabernet Sauvignore' explants more closely resembled seedlings than the parent. Lyrene (65) described two types of shoots which arose from mature laggigigm shoot tips. The first type had mature characteristics with large leaves and thick stems while the second type exhibited juvenile, filamentous stems and small leaves. The .1nuyitzg induced "juvenile" shoots proliferated vigorously and were easier to root than adult shoots or seedlings. This reversion to a juvenile state may merely be a feature of growth in litre (9, 95) although similar observations have been made with plants which were severly pruned (2h). Trippi (108) stated that asexual reproduction results in rejuvenation, and Stokes (99) reported that juvenile characteristics could be induced in coniferous species by grafting shoots from mature trees on to seedlings. Methods for propagating plants in yitzg can be divided into two main categories: 1) those which take advantage of the organized state of the primary explant and 2) those which induce an unorganized state (1, 67). The pathway taken is somewhat dependent on the inherent ability of the plant species to respond (7) although it may be possible 19 to stimulate even the most recalcitrant species if enough time and energy is spent (1, 119). Stimulating axillary shoot development from shoot tips or node segments is the most commonly used propagation procedure (1). Multiplication rates are lowest using this propagation method (77) although even modest shoot proliferation can produce large numbers of plants in one year. This is due to a decreased generation time (2). This method has the least risk of inducing aberrant plants (1, 2). With some species, it has been possible to stimulate adventitious shoot development. Since performed buds are not essential, a larger range of explants can be used. Shoot proliferation rates are generally greater using adventitious systems compared to axillary systems, however, the chance of obtaining abnormal plants is increased (77). Callus and cell suspension cultures have the greatest potential for mass cloning of plants (77, 119). This is possible at least theoretically, because each cell carries the genetic information necessary to develop into an intact plant. Plants could be recovered via organogenesis or somatic embryogenesis (119). These two types of unorganized cell cultures grow rapidly. Abbott (1, 2) reported that 20-fold increases in cell populations were possible in cell suspensions over a 10 day period. These types of cultures can be maintained indefinitely by routine subculture. Use of callus or cell suspension cultures for propagating woody plants, however, has been limited due to difficulties in obtaining shoot regeneration (1, 2, 76). These types of cultures are also subject to producing higher numbers of aberrant plants, especially from older cultures (1, 2, 105). Cytokinin(s) either singly or in combination with auxin(s) are normally required to stimulate shoot development from shoot tip or node 20 segment explants. The mode of action is still a matter of speculation (112), however, it is clear that cytokinin(s) interact with auxin(s) in the release of axillary buds from apical dominance (AZ, 8“, 112, 117). In intact plants, axillary buds can be released from apical dominance by removal of the terminal bud (12, u1, 112). Decapitation has also been used in yitzg to stimulate axillary shoot development in BQBSBLHILLLQB (86)..£runus (96)..£1zns (60),.thd9dendncn (100)..Bcsa (81, 5”) and Rubus (1n) cultures. Snir (96) reported that decapitating cherry shoots increased the shoot multiplication rate 2-3 times. The three most canmonly used cytokinins are 6-furfurylaminopurine (kinetin or Kin), 6-benzylaminopurine (BA) and 6-(Y—8’ -dimethylallylamino) purine (21p). Of these cytokinins, 21p is the most active while Kin and BA have approximately the same activity (75, 76). Cytokinin specificity, however, and not activity appears to be the most important factor in stimulating shoot proliferation. BA is the most effective of these cytokinins in stimulating shoot proliferation in yitzq and elicits responses over a wide range of genotypes including Basin; (19). Wiles. (86). Mains (61:). 12mm: (103). Rosa (no, 111) and 1m: (90). Although 21p has the highest biological activity, it is only capable of stimulating responses over a narrow range of woody genotypes. This may indicate that its mode of action and recognition site at the cellular level is more specific than that of BA. In the Eri aceae, 21p is the most effective cytokinin in stimulating shoot proliferation (6, 25, 33. 57, 63, 65). It is normally used singly at 5 to 15 mgl’1 although Anderson (6) and Strode et al. (100) reported that it was necessary to include indoleacetic acid (IAA). With other woody species, 21p has little effect. Lundergan and 21 Janick (611) compared shoot proliferation rates of M9112 cultures following treatment with BA, Kin or Zip and found that 21p was the least effective while BA.was the most effective. They did report, however, that shoots which developed on media with BA were stunted, while those on 21p appeared normal. In Boga cultures Hasegawa (91) found that 21p could substitute for BA with only a slight decrease in shoot proliferation, however, it was necessary to use 21p at 10 times the optimum BA concentration. Kinetin is the least effective of these cytokinins in stimulating shoot proliferation in woody species (86). Two reponses which appear to be common with all cytokinins are that shoot proliferation increases with increasing concentration, while shoot elongation decreases (25, 39, 59, 87, 109). Production of normal shoots is the main concern. Lane (59) reported that shoot proliferation in Ezra; cultures was the greatest at 2.0 mgl'1 BA, however, the shoots were compact and often fasciated. Normal shoots developed at a lower level of BA. The same type of response was observed in litis cultures following BA treatment (39). Vitrification is a term (27) used to describe deleterious changes in the morphology and anatomy of explants (62) as a result of environmental factors in yitgg (122). Symptoms of this phenomenon include: the development of translucent or succulent, water soaked or brittle shoots and leaves (5, 27, 33, 37, 62, 101, 122) and tissue breakdown and exudation (63). The leaves of vitreous carnation shoots are aquatic in nature (62, 122), have a higher water content and consist of spongy parenchyma cells (122), and 96-98% or 100% of the shoot tips grown on solid or liquid media respectively, lack surface waxes (101). These types of abnormal shoots usually fail to survive or root 22 following transfer from culture (5, 62, 101, 122), although Aitken et a1. (5) reported that translucent Pings radiate shoots developed normal growth if left on the same medium for several months. Fordham et al. (33) were able to root vitreous Rhododendrgg shoots which subsequently developed normally. Leshem (62) concluded that the failure of vitreous shoots to survive was due to their disorganized internal structure and loss of normal stomata. Rapid desiccation following transfer from culture may also be responsible for poor survival rates (101). Ziv et al. (122) concluded that water availability in yitzg_was the key factor responsible for vitrification. This conclusion is supported by several lines of evidence. A resumption of normal plant growth in long term cultures normally associated with dehydration of the media has been reported by several investigators (5, 62, 122). Ziv et al. (122) also observed that carnation apices cultured on agar slants developed normal, glaucous leaves while those on horizontal agar, where water accunulated were vitreous. High relative humidity in litre has also been shown to cause vitreous leaf and shoot development (122). This was postulated by others (5, 101), but not supported. In culture, the relative hunidity often approaches 1001 (111, 122). This condition stimulates rapid, succulent growth and shoot proliferation, and may lead to the development of vitreous shoots. Ziv et al. (122) were able to increase the percent of normal carnation plantlets by reducing the relative humidity ;g_vitr , and these plants were more capable of surviving transfer from culture than vitreous plants. Another factor influencing vitrification is the water potential of the culture media (27, 62). Debergh et a1. (27) measured the water potentials of media supplemented with varying agar, sucrose or mannitol 23 concentrations and found that increasing these supplements resulted in more negative water potentials. They were able to show that agar was responsible for the metric component of the media water potential and sucrose or manitol were responsible for the osmotic component. Interestingly, increasing the agar conentration was the only way to reduce vitrification. Increasing the sucrose or mannitol concentration had no effect. This can be explained by the fact that sucrose and mannitol are capable of penetrating tissue while agar cannot (27). Movement of sucrose or mannitol into the explants changes the osmotic balance between the tissue and medium and causes water to move into the tissue. Sucrose in culture is both a carbon source and osmoticum (16). It also plays an important role in creating the critical turgor pressure required for cell expansion and shoot formation. In this respect, mannitol can substitute for sucrose but agar cannot. Fortunately, agar behaves in a different manner. Various hypotheses have been presented to explain the way agar effects shoot proliferation and vitrification but the most plausible is that increasing the the agar concentration creates water stress (62) in the tissue and makes it more difficult to attain the critical turgor required for growth (16). Other explanations for the role of agar include: an effect on the diffusion rate of molecules (88), with particular emphasis on cytokinins (27); the presence of an inhibitor or toxic substance in the agar (82, 88) and the inhibitory effect of an excessively hard gel (76). In an interesting study, Singha (88) reported that agar played an important role in controlling shoot proliferation in cultures of M313: 'Almey' and £123; 'Seckel', but in different ways. Shoot proliferation in the Helga cultures was the greatest at 0.31 agar and decreased at 2“ higher concentrations. Cultures of gyggg, however, responded poorly at O or 0.3% agar with increased shoot proliferation only at higher agar concentrations. Singha (88) was somewhat at a loss to explain these results but it seems likely that these species respond differently to the availability of water. Although increasing the agar concentration reduces vitrification and improves shoot quality it also decreases shoot proliferation (16, 27). Debergh et al. (27) observed that increasing the agar concentration in cultures of Elgar; egglyggg, Globe Artichoke, to 1.1% eliminated vitrification and greatly reduced shoot proliferation. This is a cause of concern because it may increase the cost of producing plants in 11539. However, if preventing vitrification increases the nunber of usable shoots then the control measure must be accepted. Returning intact plants to their natural envirOnment is the main objective of stage 3. This includes: developing an effecient rooting protocol, determining procedures for acclimating plants to ambient conditions and evaluating plants produced in litre. Auxins are normally required to stimulate root development ingzitng (79). The three auxins used most often are: indoleacetic acid (IAA). indolebutyric acid (IBA) and naphthaleneacetic acid (NAA). These are listed in order of increasing physiological activity (76). Hasagawa (#1) studied the effect of IAA, IBA and NAA on the rooting of Rosa hybrids shoots. He found that IAA and NAA at 1.0 mgl‘1 and 0.03 or 0.1 mgl'1 respectively were more effective than IBA. In a separate study, Snir (96) observed that 100% of Prague ayiyg_shoots rooted on NAA media following a 111 day culture period compared to 30% on IBA, however, if left for a longer period of time all shoots eventually rooted. 25 An undesirable side effect of NAA is that it often stimulates callus development (59, 87). Lane (59) reported that concentrations of NAA above 0.1 mgl‘1 stimulated callus growth and reduced rooting of .Bzunng and §pirga_microcuttings. Lowering the salt concentrations in MS media to 1/A or 1/2 stimulates rooting in some figsageggs species (#1, SA, 59). Hyndman et al. (86) concluded that this stimulatory effect was mainly due to the reduction in nitrogen concentraion of the media. Light has been shown to have a negative effect on rooting of some Rosaceogs species (38, 79). Norton and Boo (79) suggested that this was due to the accumulation of endogenous IAA in the dark. Deleting agar from the rooting medium has been shown to stimulate rooting of 9:9111122 (10), Prunus and Spires (59) and,iitia (39) shoots. No reasons were given by these authors for the inhibitory effect of agar, but it may be due to its effect on the water potential of the media or to the presence of toxic substances in the agar (82). Other procedures which stimulate root formation in yitzg include: dipping flalgg shoots in 0.1 mgl'1 IBA and culturing them in an inverted position (3) and supplementing the rooting media with 162 mgl" phloroglucinol (88, ”9). With some species (33, 35, 63, 65, 66, 69), it has been possible to root microcuttings under septic conditions. To prevent desiccation the microcuttings must be placed in a high humidity chamber or under mist. It is interesting, that in most of these instances no hormone treatments were required to stimulate rooting. This was true even in species which are normally difficult to root (69). McCown and Amos (69) suggested that this was due to a change in the physiological state of the micropropagated shoots. Lyrene (66) concluded that the ease of 26 rooting was due to the phenomenon of rejuvenation. Evaluation of woody plant populations propagated in litre has been limited, partly due to the small numbers of plants propagated. Other problems which have undoubtedly limited evaluations are: space limitations, the extended period of time to grow plants to maturity and the use of seedling explants, where some segregation would naturally be expected. Zimerman (121) suggested that tissue cultured plants should be evaluated by their phenotypic appearance and field performance. Swartz et al. (102) successfully used these criteria to evaluate clones of .Ezggania x engages; cultivars. With few exceptions, they observed that the phenotypes of the clones were the same as plants propagated by standard techniques. The tissue culture clones did, however, exhibit some increased vigor and axillary bud activity. These differences may be due to epigenetic or phase changes (102, 121). Krul and Myerson's (56) observations with 1131§,'Seyval' support this argument. They observed that vines regenerated from callus by somatic embryogenesis were phenotypically identical but more closely resembled the original description of the cultivar than the parent plants. m The phenotype of a plant is controlled by its genotype and growth environment. This applies to explants in culture just as it does to plants in nature. The in yitgg culture environment, however, is more closely controlled and can be easily manipulated by changing various factors including plant growth regulators. Establishing aseptic, actively growing cultures can be a difficult 27 task depending upon plant species, condition of the plant material and time of year. Two problems which must be overcome are: surface contamination and phenolic secretion. Stimulating rapid shoot proliferation in 12322 is genotype and growth regulator dependent. Some genotypes respond more readily than others, with differences sometimes apparent even between closely related cultivars. Some species have been shown to become more responsive with increasing time in culture. This may be due to the phenomenom of rejuvenation. Plants recovered from these cultures often exhibit juvenile morphological characters. Media development is an empherical process requiring a screening of components. In the future, it may be possible to select the appropriate media by analyzing endogenous hormone levels. This technology, however, still remains to be developed. As noted earlier, BA is the most stimulatory cytokinin for promoting shoot proliferation in cultures of woody species. Auxins are generally required to induce root development in 11.11.29,. although this is not always the case. With some species this is a simple task, however, for the majority this requires some effort. Two areas which require additional emphasis are: developing procedures for acclimating and evaluating plants on a commercial scale. Micropropagation of woody species is now in its infancy but with research concentrated on the areas previoulsy mentioned, should grow dramatically in the near future. MATERIALS AND METHODS .Cnltuna.lniiiaticn Actively growing shoot tips, 7-10 cm, were removed during the spring growth period from flowering 6-8 year-old trees of Age; rubrum 'Red Sunset', growing at Beaumont Nursery, Michigan State University, East Lansing, Michigan. The shoot tips were cut into single node segments and shoot tips, 3-8 cm, the expanded leaves were removed and the explants were immersed in cold running tap water for 5 minutes. The explants were surface sterilized by immersion in a 101 Clorox solution plus 0.051 Tween 20 for 30 minutes followed by 5 rinses with sterile distilled water. The last rinse was decanted and the explants were held in a covered glass bowl until being prepared for culture. The shoot tips and node segments, 3-fl cm, were trimmed at the base and placed on a slant in 100 x 15 mm Petri @dishes, 5 shoot tips or node segments/dish. The Parafilm<:)sealed dishes received a photon flux fluence rate of 3apa m‘zs“1 (0.3. F96T120W, n00-700nm) during a 16 hr photoperiod at 25 1 2° C. The culture medium was a modified Linsmaier and Skoog (LS) (3) with thiamine RC1 1.0, myo inositol 100, sucrose 30,000, agar (Sigma grade IV) 8000 mgl'1 and pH 5.8. The medium was autoclaved at 15 lb in‘2 (1035 kPa) for 20 minutes, cooled to u5° c and dispensed into 100 x 15 mm Petri<:>dishes, 25 ml/dish. To control phenolic secretion, shoot tips 3-A cm, were: a) soaked in filter sterilized (O.A5Km Millipore filter) citric acid, 0, 150 or 28 29 300 mgl“ for 30 minutes following surface sterilization and then held in a humid environment until being placed in culture or b) transferred to fresh LS medium with O or 1.0 [mgl‘1] 6(1-K-dimethylallylamino)- purine (21p), 6-benzy1aminopurine (BA) or 6-furfurylaminopurine (Kin) at 3 day intervals. Partially damaged explants were retrimmed to remove the injured tissue and returned to culture and dead or contaminated explants were discarded. After 3 or A transfers, actively growing shoots which had stopped secreting phenols were transferred to shoot proliferation median. Shoct.£rcliferati2n In a preliminary experiment, axillary shoot proliferation was observed on A,.ngpnpm 'Red Sunset' shoot tips after a 6 week culture period on LS + 10 mgl'1 21p. Proliferating stock cultures were established by culturing actively growing shoot tips on this median for one or more culture periods. Shoot tips, 1.5 cm, from these cultures were used to initiate shoot proliferation and vitrification experiments. Except where noted, shoot tips were cultured singly for A wks in 60 ml bottles, then transferred intact to 280 ml bottles for 2 wks. To determine the effect of cytokinins on shoot proliferation, shoot tips were placed on LS + 0, 1.0, 5.0, 10.0, or 15 mgl ‘1 21p, BA or Kin. Shoot tips were also placed on LS + 10 mgl'1 21p supplemented with 0, 50, 100, or 200 ml”1 adenine sulfate or casein hydrolysate. In a separate set of experiments randomly selected shoot tips, 1.5 cm, were grown for A wks in 60 ml bottles on LS + 10.0 mgl-1 21p, decapitated 2 nodes above the basal callus mass and returned to 60 ml bottles. Shoots were harvested from the decapitated cultures at 10 day 30 intervals. Shoot tips, 1.5 cm, were placed on LS + 10.0 mgl'1 zip, with the following modifications or treatments to determine their effect on shoot proliferation and vitrification: a) sucrose 10, 20, 30, A0, or 50 gl“, b) agar A, 6, 8, 10, or 12 gl", c) salt concentration 25, 50, 75, or 1001, d) temperature 20° c for 0, 1, 2, 3, or u wks followed by 25° 0 for a total of 6 wks, e) comparison of 25 x 150 mm culture tubes cultured on a slant with 60 ml bottles, n wk culture period and f) comparison of 60 and 280 ml bottles; shoot tips were grown for u wks, decapitated 2 nodes above the basal callus and returned to the same size culture vessel for 2 wks. Three culture vessels, 60 and 2&0 ml glass bottles with metal screw caps and 25 x 150 m1 glass culture tubes capped with plastic KaputsCED were used in the experiments with 25, 50 and 25 ml media/vessel respectively. The media were heated to dissolve the agar, dispensed into the culture vessels and autoclaved as described previously. One shoot tip was placed in each culture vessel with 5 or 10 replications/treatment. The experiments were arranged in a completely randomized design and were repeated at least twice. Data were analyzed using the F test with means separated using Duncan's Multiple Range Test or Student's T test (2351). Results from duplicate experiments were combined unless there were significant differences, then the data were presented separately . Rootin , Acclimatign and Egaluatign To determine the effect of auxins on root development, shoot tips, 1.5 cm, from aseptic stock cultures were placed on LS + O, 0.01, 0.1 or 31 1.0 mgl" indolebutyric acid (IBA), naphthaleneacetic acid (NAA) or indoleacetic acid (IAA) for 2 wks. Each treatment was replicated 10 times and the experiments were repeated at least twice. Experiments were designed and the data analyzed as described previously. One shoot tip was placed in each 25 x 150 mm glass culture tube with 25 ml media/culture tube. All components of the media were 'autoclaved except for IAA which was filter sterilized (0.A5/tm Millipore filter) and added to cooled (A5o C) media. Shoot tips were also rooted in 0.95 1 bottles, 50 shoot tips/bottle. Each bottle contained 75 ml LS basal medium and the aluminum foil sealed bottles were placed in a horizontal position. 2 1 All cultures recieved a photon flux fluence rate of 3A/LE m' s' (G.E. F96T12CW, AGO-700 am) under a 16 hr photoperiod at 25 :_2 C. Rooted shoots were removed from culture, washed in tap water to remove the agar and planted in AC-6/12 Cell Paks(:>(Ball Seed 00., West Chicago, Illinois) using a peat-lite planting mix (VSP, Bay Houston Towing Co.). The Cell Paks<:>were placed inside plastic bags to prevent dessication and were held in a growth room at a photon flux fluence rate of 38-6A/4E m‘2 s"1 (G.E. F96T12CW, ADO-700nm) under a 16 hr photoperiod at 25:2 C. The plants were acclimated to the ambient environment by gradually opening the plastic bags over a 2 wk period and were then transferred to the greenhouse. Plants in the greenhouse were grown under a natural photoperiod supplemented with a A hr night break, 10:00 p.m.-2:00 a.m. of 21.5/uE ln'"2 s'1 (G.E. 100w incandescent) from September 15-March 15. The day and night temperature was 230 C minimum and the day temperature fluctuated higher with the season. Plants were fertilized once/week with 150 ppm N, 20-20-20 using acidfied (HBPOA) water to maintain the 32 planting medium at pH 6.5. After 2 wks, the plants were transferred to 3.8 1 plastic pots using the same planting mix. Plant height was measured biweekly and the plants were evaluated visually. RESULTS The establishment of aseptic, actively growing cultures of A; .nnpznn 'Red Sunset' was hindered by surface contamination and the secretion of polyophencls. Explants collected in the spring during the first growth flush were readily surface sterilized; whereas, those collected later in the year were increasingly more difficult. Surface contaminants could be eliminated but the concentration of sterilant required also seriously damaged or killed the shoot tip or node segment explants. Injured explants secreted phenols from the wounded surfaces eventually resulting in death of the affected tissue (Fig. 1). This was accompanied by browning of the surrounding median. The only effective method found for controlling phenolic secretion was the use of a preconditioning treatment. This treatment consisted of subculturing undamaged or partially damaged, 3-A cm shoot tips or node segments to fresh LS basal median at 3-A day intervals. Damaged tissues and dead or contaminated explants were discarded. Addition of 1.0 mgl'1 Zip or BA to the preconditioning medium stimulated basal callus and new shoot growth. In this respect BA was more effective than 21p. Kinetin at this concentration was not effective in promoting callus or shoot growth. After 3-A subcultures the phenolic secretion would cease and the explants could be transferred to Stage 2 experimental media. Attempts to control phenolic secretions were also made by soaking explants in 150 or 300 mgl"1 citric acid for 30 minutes following surface sterilization. However, this treatment resulted in further 33 .3” Figure 1. Phenolic secertion from Age; rubrum 'Red Sunset' node segments. 35 .— mesa: 36 injury. Improved explant survival could be obtained by holding the shoot tips or node segments in a covered glass bowl before dissection rather than floating in the last water rinse. Shootizrclifszaiicn The morphogenetic responses of A, rubrum 'Red Sunset' shoot tips to 21p, BA and Kinetic are shown in Table 2. BA was the most consistent of the three cytokinins tested in stimulating shoot proliferation with 5.9 shoots/culture at 5.0 mgl“, although 21p was nearly as effective with 3.0 shoots/culture at 10.0 mgl". With both of these cytokinins, axillary bud break was observed during the third or fourth week of culture (Fig. 2A) followed by shoot elongation during the next two week culture period (Fig. 2B). Axillary bud break was also observed at the lowest node of shoot tips cultured on 10.0 mgl" Kinetin, but subsequent shoot elongation did not occur. Adventitious shoot development was not observed with any cytokinin treatment. The requirement for a high level of cytokinin to stimulate axillary shoot development in 'Red Sunset' cultures can be seen in Fig. 3. Shoots placed on LS medium lacking cytokinins rapidly developed roots and grew slowly with little or no shoot elongation during a A week culture period. In comparison, shoot tips placed on LS + 10.0 mgl"1 21p elongated and axillary bud break occurred during the same period of time. Mean shoot length (Table 2) varied significantly depending on cytokinin concentration. With 21p and BA, shoot length was the greatest at the concentration just below the optimum for shoot proliferation. 21p and BA stimulated large amounts of callus a the base of the 37 Table 2. The morphogenetic responses of Age; ggbggm_'Red Sunset' shoot tips after 6 wks culture in LS medium containing the cytokinins, 2ip, BA and kinetin. Cytokinin Shoot Mean Shoot Damaged Mean Shoot Mean Callus (mgl'1) Proliferation Number" Shoots Length Fresh Wt. 1 1 (cm)x (gm)x 21p 0 0 1.0 b 0 1.0 _ 0 1.0 b 0 1.9 b 0.53 b 5.0 20 1.3 b 0 6.1 a 3.02 a 10.0 50 3.0 a 33 3.3 b 3.21 a 15.0 10 1.1 b 9 1.9 b 0.71 b BA 0 0 1.0 b 0 1.0 70 2.A b 8 A.5 a 0.81 b 5.0 80 5.9 a 25 2.6 b 3.01 a 10.0 30 1.A b 7 1.5 bc 1.31 b 15.0 10 1.1 b 0 1.3 c 0.55 b Kin 0 0 1.0 a 0 1.0 0 1.0 a 0 1.6 b 0.10 b 5.0 10 1.1 a 0 2.A b 0.A3 a 10.0 0 1.0 a 0 1.9 ab 0.A2 a 15.0 0 0.8 a 0 1.1 c 0.36 a xMeans for a given cytokinin and column with the same letter are not significantly different. Duncans Multiple range (P551). 38 Figure 2. Acer rubrum 'Red Sunset' shoot tips. A. Axillary bud break after 11 wks on LS + 10.9 mgl'1 zip. B. Axillary shoot elongation after 6 wks on LS + 10.0 mgl‘ 21p. A0 Figure 3. Growth of Agar Mm 'Red Sunset" shoot tips after A wks on LS + 10.0 mgl" 21p (left) or LS basal median (right). .m 0.5m: A2 shoot tip explants. Callus fresh weight varied significantly depending on the cytokinin concentration. The callus was compact, nodular and whitish- or reddish-green. Vitreous shoots were observed in several of the 21p and BA treatments, with substantial damage occurring at the concentrations optimum for shoot proliferation. Symptoms of vitrification included: translucent leaves and shoot tips; succulent, water soaked tissues and guttation from leaves and shoot tips; followed by death of the shoot tips and tissue collapse. In some cases, stem swelling, compact internodes and intumescences from lenticels were also observed. Shoots with vitreous symptoms did not survive transfer to fresh medium. Damage normally occurred first at the apex of the primary shoot and proceeded toward the base. Death of the shoot terminals often stimulated shoot proliferation. However, if left unattended, entire cultures would succumb to these symptoms. In some instances, normal growth would resume if cultures were held for eight to ten weeks without subculturing. Decapitating A..nnhnyn 'Red Sunset' shoot tips stimulated axillary shoot proliferation (Table 3 and Fig. A). Shoot proliferation was more consistent using this procedure than when shoot tips were left intact. The terminal shoot tips could be returned to fresh medium to reinitiate the shoot proliferation cycle. Shoots, 1.0 cm or larger could be harvested 10 days later. The number of vitreous shoots, however, increased at the second harvest date. Harvesting shoots at intervals greater than 10 days also increased the number of damaged shoots. These shoots were succulent and tender and easily damaged by adverse conditions. Fasciated shoots and shoots with 3 leaves/node were occasionally ”3 Table 3. The morphogenetic responses of Ager.ngggn 'Red Sunset' shoot tips to decapitation. The shoot tips were cultured for A wks on L8 + 10.0 mgl"1 21p, decapitated and basal sections were returned to the same median with shoots harvested at 10 day intervals. Nanber Shoot Mean Shoot Nanber Damaged Shoots Cultures Proliferation Harvest (1) (1) 1 2 30 97 3.8 1 15 100 5.7 0 11 100 5.7 5.3 5 17 15 100 5.7 6.5 0 0 AA Figure A. .Apennrubrum 'Red Sunset' shoot tip explants 2 wks after decapitation, A.0 shoots/exalant. The shoot tips were cultured for A wks on LS + 10.0 mgl' 21p, decapitated and returned to the same median for 2 wks. .v scam: A6 observed in the decapitated cultures. These abnormal shoots were easily identified and removed. The morphogenetic responses of 'Red Sunset' shoot tips to media amended with adenine sulfate or casein hydrosylate are shown in Table A. The mean shoot number was significantly increased by the addition of 100 mgl'1 adenine sulfate to the culture medium compared to the control, LS + 10.0 mgl'1 21p, while 200 ng.‘1 had a toxic effect. Mean callus fresh wt. decreased significantly at adenine sulfate concentrations above 50 mg1'1. Significant experiment by treatment interactions in mean callus fresh weight were observed between the three adenine sulfate experiments (Fig. 5). Casein hydrosylate, at the levels tested, had no effect on the growth of LM 'Red Sunset' shoot tips. .SDQQL.22211£2£§&120.EQQHIILEAQAQELAQQ .Aggnnngzpm 'Red Sunset' shoot tips responded differentially to sucrose or agar concentrations (Table 5). The percent shoot proliferation and mean shoot number increased with increasing sucrose concentration although there was no significant difference between 30, A0 or 50 gl'1. Culturing shoots on 10 gl"1 sucrose severely restricted shoot proliferation, shoot elongation and callus growth. Increasing the sucrose concentration above 30 gl‘1 did not reduce the incidence of vitreous shoots. Callus fresh weight increased significantly with increasing sucrose concentration. As the sucrose concentration of the LS + 10.0 mgl"1 21p medium was increased the amount of anthocyanin in the shoot tip explants increased A7 Table A. The morphogenetic responses of Age: rm ' ed Sunset' shoot tips after a six week culture period in LS + 10.0 mgl‘ 21p and adenine sulfate or casein bydrolysate. Supplement Shoot Mean Shoot Damaged Mean Shoot Mean Callus (mgl'1) Proliferation Numberx Shoots Length Fresh Wt. (1) (1) (cm)x (gm)x Adenine sulfate 0 53 2.1 bc 6 A.59 a A.02 a 50 53 3.7 ab 11 A.A8 a 3.91 a 100 67 A.A a 1A 3.81a 2.73 b 200 A0 1.A c 0 3.A8 a 0.97 c Casein hydrosylate 0 A7 2.8 a 1A 3.8A a A.2A a 50 A7 2.9 a 20 3.67 a 3.83 a 100 A0 2.3 a 17 3.21 a 3.51 a 200 A0 2.A a 22 2.96 a 3.32 a xMeans for a given supplement and column with the same letter are not significanlty different. Duncans Multiple Range (2551). 48 To 0 - c 6.0 - ..."O.oco e e o c A e a V 5 . O b ... 4-) o G.) O 3 0. fi 11. 0 - 3. g" e a. 1; g \ '- {—1-1-1 \\ o .' s o 8 3. O b \X" “ O. a a" \ \‘ e. o \ fi 0 a) o' ‘t \ '. E 0' \ \ o s‘ \ '. ‘s \ .0 2 . 0 . ‘. \\ '. \ \ ‘. e‘ \ o c \ s‘ \ s‘ \\ s l . O ‘- ‘e \ s \ $ \ s s ‘0 l I | L O 50 100 200 Adenine Sulfate (mgl-l) Figure 5. The effect of adenine sulfate on basal callus formation in Acerrubrum 'Red Sunset' shoot tip cultures after 6 wks on LS + 10.0 mgl 2ip. Significant experiment by adenine sulfate concentration interactions for mean callus fresh weight were observed between the three experiments: l-"-, 2000and 3---. R9 Table 5. The morphogenetic responses of Acer rubrum 'Red Sunset' shoot tips after a six wk culture period in LS + 10.0 mgi" zip and varying sucrose or agar concentrations. Treatment Shoot Mean Shoot Damaged Mean Shoot Mean Callus (81' ) Proliferation Numberx Shoots Length Fresh Ht. (1) (1) (cm)x (3m)x Sucrose 10 0 1.0 c 0 1.70 c 0.17 d 20 20 1.” be 0 3.89 a 1.23 c 30y 60 3.7 ab 16 3.18 b 3.30 b no 80 3.5 ab 11 ”.81 a u.u3 a 50 100 n.2 a 33 3.83 ab “.3” a 383? u 30 2.3 b 22 u.u3 a 2.29 b 6 30 1.8 b 6 3.39 ab 2.10 b BY 60 u.8 a 19 3.03 bc 2.7a ab 10 30 1.“ b 7 2.37 be 3.16 a' 12 20 1.3 b 0 1.99 c 2.90 ab 1‘Means for a given treatment and colunn with the same letter are not significantly different. Duncans Multiple range (P551). yControl treatment. 50 and was highly visible at 50 g1‘1. Stems, petioles and leaves were reddish-green and some of the new shoots, especially those from lower nodes, were entirely red. Shoot proliferation was most consistent at 8 gl‘1 agar and the mean shoot nunber was significantly lower at agar concentrations below or above 8 g1". Vitreous shoots were eliminated by raising the agar concentration to 12 gl“. Mean shoot length decreased significantly with increasing agar concentration. A highly significant experiment by treatment interaction in callus fresh weight was observed between the two agar experiments (Fig. 6). Callus fresh weight was essentially the same at 11 or 6 gl‘1 and varied significantly at higher concentrations. Lowering the salt concentrations of LS + 10.0 mgl" zip medium by 25% significantly reduced shoot proliferation and meanwshoot nunber (Table 6). Shoot length and callus fresh weight were also decreased by lowering the salt concentration but the reduction as gradual. Decreasing the salt level had the least effect on callus fresh weight. Holding A... mgr-um 'Red Sunset' shoot tips at 20° c for at least two weeks before transfer to 25° C significantly reduced shoot proliferation, mean shoot nunber and callus fresh weight. Increasing the length of the 20° C treatment to 3 or 11 weeks had a deleterious effect on all parameters. A significant experiment by temperature interaction was observed in mean shoot nunber between the two experiments (Fig. 7). 51 5'0 ‘F ' 14.0 -- | E) 12’ 00 "a . 3 3.0 1. .c: U) 0) $4 EL. I . g \ I Q o— I 8 2.0 ‘- “" C O o 4 / (1) 2 O 1.0" l I I l I ‘ h 6 8 10 12 Agar (gl-1) Figure 6. The effect of varying agar concentrations on basal callus formation in Ager rubrum ’Red Sunset' shoot tip cultures {after 6 wks on LS + 10.0 mgl 2ip. A highly significant experiment by agar concen- tration interaction for mean callus fresh weight was observed between experiment: 1 (0) and 2 (I). 52 Table 6. The morphogenetic responses of Age: gypggm,'Red Sunset' shoot tips to variable salt con ntrations or temperature regimes. Basal medium was LS + 10.0 mgl' 21p. Shoot tips in the temperature experiment were held for 0 - ” wks at 200 C then transferred to 250 C. Data were taken after a 6 wk culture period. Treatment Shoot Mean Shoot Damaged Mean Shoot Mean Callus Proliferation Number Shoots Length Fresh Wt. (1) (1) um)" (3:11)" Salt Canon. (1) 100y 80 ”.7 a ” ”.92 a 3.”” a 75 0 1.0 b 0 3.93 a 3.2” a 50 20 1.1 b 0 1.93 b 2.”0 a 25 10 0.9 b 11 1.1” b 0.50 b Temperature 200 C wks 03 80 5.0 a 26 ”.17 a 3.25 a 1 70 3.9 ab 26 ”.20 a 3.0” a 2 50 2.3 bc ”3 3.31 ab 1.8” b 3 20 1.9 be 11 2.1” b 1.32 bc ” 0 1.0 c 0 2.23 b 0.93 c 1‘Means for a given treatment and colunn with the same letter are not significantly different. Duncans Multiple range (P555). yControl treatment. 53 Mean shoot number and shoot length in 25 x 150 mm culture tubes and 60 ml bottles were not significantly different as analyzed by Student's t test (P551) (Table 7). However, explant quality was better in 60 ml bottles than in the culture tubes. Leaves and shoots in 60 ml bottles were green and appeared normal while many of those in the culture tubes had a waterlogged, translucent appearance. Shoot tips in the culture tubes had a tendency to grow upwards against the glass where_water condensed. This situation often led to death and collapse of the shoot tips. Death of the shoot terminals enhanced axillary bud break and shoot elongation. The gain in shoot proliferation, however, was negated by the spread of vitreous symptoms to the new growth. After a four week culture period in 60 or 2”0 ml bottles, there was a marked difference in the growth of 'Red Sunset' shoot tips. Axillary shoot elongation was evident in 501 of the cultures in 60 ml bottles compared to 10$ in 2”0 ml bottles. New shoot growth was evident in both culture vessels but internodes and petioles were more compact in 2”0 ml bottles. Explants growing in 2”0 ml bottles had a hardened appearance. The same trends in shoot proliferation and elongation were evident following decapitation (Table 7). Mean shoot length was significantly reduced in 2”0 ml bottles compared to 60 ml bottles. The number of damaged shoots after the six week culture period was lower in 60 ml than 2”0 ml bottles. Mean callus fresh weight was significantly greater in the 2”0 ml bottles than in the 60 ml bottles. 5” 8.0 7.0 ’ 6.0" 5.0" ”.0 - Mean Shoot Number 3.0 ' 2.0. \ 1.0' = 5 o l 2 3 h 0 Weeks (20 C) ' Figure 7. The effect of varying temperature regimes on axillary shoot proliferation in cultures of Acer rubrum 'Red Sunset'. Shoot tip ex- plants were held for 0—” wks at 2OUC followed by t ansfer to 250C for a total of 6 wks. Basal medium was LS + 10.0 mgl- 2ip. A significant experiment by temperature interaction in mean shoot number was observed between experiment: 1 (°) and 2 (I). 55 Table 7. The morphogenetic responses of Aggn.ggb£gn_'Red Sunset' shoot tips in different culture vessels. Shoot tips were cultured in LS + 10.0 mgl‘“1 2ip. In the six wk experiment, shoot tips were cultured intact for four wks, then decapitated two nodes above the base and the basal mass was returned to the same culture vessel for two weeks. Culture Shoot Mean Shoot Damaged Mean Shoot Mean Callus Vessel Proliferation Nanber Shoots Length Fresh Ht. (1) (1) (cm)x (gm) x Four Weeks 25 x 150 mm Culture Tubes ”5 2.8 27 3.01 60 ml Bottles 30 1.8 0 3.”6 .____ Six Weeks 60 ml Bottles 100 ”.7 u 3.0n" 3.3a auo ml Bottles 50 2.7 30 1.35 5.88.. xStudents' t test (2351). so mmmm 'Red Sunset' shoot tips rooted readily during a two week culture period on LS basal medium or L8 supplemented with IBA, NAA or IAA (Table 8). Roots were visible in all treatments 5-6 days after the shoot tips were placed in culture. The roots generally arose from the base of the shoot tips without intervening callus. The adventitious roots were white and appeared normal (Figure 8), although there were no secondary roots or root hairs. Raising the concentrations of IBA or NAA to 1.0 mgl", significantly increased the mean root number and decreased root length. At this concentration, these auxins stimulated callus formation which was quickly followed by root development. Roots were observed at the base of the shoot tips on 1.0 mgl" NAA after 3 days culture. The roots were short, abnormally thickened and brittle. Little or no new shoot growth was observed following transfer of shoots to rooting media. The internodes were compact and the leaves were green or dark-green. 'Red Sunset' shoots also rooted with 100$ efficiency in 0.95 liter bottles. ,Agg;,£gbggn_'Red Sunset' plants growing in the green house were identical (Fig. 9) with the exception of occasional plants with 3 leaves/node. This was true even of plants from cultures 2.5 years-old. The plants grow rapidly following transfer from culture (Fig. 10). The leaves were 5 lobed and heart shaped at the base. This is in contrast to leaves of parent plants which were generally 3 lobed and rounded at the base. After 18 weeks the plants had reached a height of 82.9 cm. Rapid growth was also observed in containers under field conditions with adequate nutrition. 57 Table 8. The effect of the auxins, IBA, NAA or IAA incorporated in LS medium on rooting of Ager ruprgm 'Red Sunset' shoot tips after a 2 wk culture period. Auxin Rooting Mean Root Mean Root (mg1'1) (3) Numberx Length mm)" IBA 0 85 2.” b 2.30 a 0.01 100 2.7 b 2.37 a 0.1 100 3.3 b 2.01 a 1.0 95 10.” a 0.72 b NAA 0 100 2.7 b 2.78 a 0.01 100 2.6 b 2.26 a 0.1 100 3.6 b 1.81 b 1.0 95 6.” a 0.”3 c IAA 0 100 3.0 a 2.15 a 0.01 100 ”.0 a 1.90 ab 0.1 100 3.3 a 2.37 a 1.0 100 ”.0 a 1.”? b xMeans for a given auxin and column with the same letter are not significantly different. Duncans Multiple Range (2551). 58 Figure 8. Rooted A99: rubrum 'Red Sunset' shoot tips 2 wks after being placed on LS basal medium. .m elem; 60 Figure 9. Ace: w 'Red Sunset' plants propagated in vitro; ” wks after transfer from cultures to planting median, height ”.1 cm. chasm: 62 100 - 90 ' e 80 ' '2 70- O .p g 60- -.-1 a) :1: +3 50!- 5 H Q. g to ' <1) 22 o I I l l l I l l l 6 8 lo 12 1h 16 18 20 Weeks Figure 10. Growth rate of in_vitro propagated Acer rubrum 'Red Sunset' plants in the greenhouse. Four weeks after transfer from culture, uni- form plants were transplanted in 3.8 liter plastic pots using a peat- lite planting mix (VSP, Bay Houston Towing Co.). The plants were ferti- lized once/week with 150 ppm N, 20-20-20 during the growing period from ”/13/82-8/23/82. 63 Plantlets transferred to the greenhouse during November, December and January grew poorly and often became dormant. The growth rate of older plants also slowed during this period of time and most of the plants became dormant. DISCUSSION The most difficult stage in propagating Apex; m ' Red Sunset' jg; .yitrg,was the initiation of aseptic, actively growing cultures. As the name of this cultivar implies, a large amount of anthocyanin is produced in the tissues. This red coloration is particularly evident on new stem growth, flowers and fruit during the spring and on leaves during the fall. Surface sterilization of actively growing 'Red Sunset' shoot tips or node segments with a diluted Clorox solution injured the tissues and stimulated phenolic secretions. This is suggested because after several subcultures phenolic secretion ceased and did not-reappear even upon dissection. Damaged explants rapidly turned brown and died if left unattended. The secretion of phenols from 'Red Sunset' explants was not entirely suprising, however, since anthocyanins and phenols share a common biosynthetic pathway (95). Similar difficulties were also encountered with attempts to establish cultures of A; gignala in gitzg (Meyer and Welsh, 1982 unpublished data). Shoot tips of this species collected during the spring growth flush are bright red and rapidly turning black following surface sterilization. Phenolic secretion in 'Red Sunset' cultures could be controlled by using a preconditioning treatment (22). This treatment consisted of placing shoot tip or node segment explants, 3-” cm, on LS + 10.0 mgl'1 BA or 21p and transferring at 3-” day intervals. These two cytokinins stimulated new shoot growth and also callusing of wound surfaces. Rapid subculturing allowed removal of damaged and dead tissue or contaminated 6” 65 explants. Similar procedures have been used by others (1”, 67) to effectively control phenolic secretion. Holding 'Red Sunset' explants in a humid environment prior to dissection also helped to reduce injury by polyphenols. In some instances (7, 67), soaking explants in antioxidants or incorporating them in the culture medium has proven effective in controlling phenolic secretion. This was not observed, however,‘with.AL .ruhrgn 'Red Sunset'. Soaking 'Red Sunset' shoot tips or node segments in citric acid resulted in further injury. Broome and Zimmerman (1”) made a similar observation with Rubus cultures. These responses may indicate that damage due to phenolic secretion.was only secondary to damage caused by the surface sterilants. Damage control, therefore, should be aimed at modifying the surface sterilization procedure rather than at controlling end products. High levels of BA or Zip, 5.0 or 10.0 mgl.1 respectively, were required to overcome apical dominance in AL zgpggm 'Red Sunset' cultures. At these concentrations, axillary shoot proliferation only occurred after the formation of a basal callus mass and elongation, of the shoot tip explants. Lower levels of BA, 1.0 mgl"1 or Zip, 1.0 or 5.0 mgl"1 also stimulated basal callus formation and shoot elongation but shoot proliferation rates were significantly reduced. Both the high cytokinin requirement and the sequential growth pattern imply the presence of an endogenous hormone gradient in 'Red Sunset' shoot tips. Fari and Czako (32) made a similar observation with gapsiggnqgnngum 'T. Hatvani' hypocotyl sections. They reported that shoot development was dependent on the position of the explants along the hypocotyl. Explants taken Just below the cotyledons developed shoots while those closer to the root developed callus or roots. They concluded that differentiation 66 was controlled by the complement of exogenous and endogenous growth substances. This may also be the case with 'Red Sunset' shoot tip cultures. To stimulate axillary shoot development, the cytokinin levels in the medium had to be above a critical level. Since auxin(s) and cytokinin(s) interact in regulating the growth of axillary buds (61, 117), one possible explanation for these observations is that Ag.znnnum shoots produce a high level of endogenous auxin(s). This hypothesis is supported by several lines of indirect evidence: 1) young L m trees have a pyramidal growth habit (28) suggesting strong apical dominance, 2) shoot proliferation in yit§g_was most consistent when shoots on 10.0 mgl”1 21p were decapitated and 3) 'Red Sunset' shoots rooted readily in Film on basal median lacking auxins. The most effective cytokinin in stimulating axillary shoot proliferation in 'Red Sunset' cultures was BA. This appears to be a general trend with many woody species (19, ”0, ”1, 6”, 86, 90, 103). It was also possible to stimulate axillary shoot proliferation using 21p but twice the optimum BA concentration.was required. Haseqawa (”1) made a similar observation with 39;; mg; cultures where zip at 10.0 mgl“1 was nearly as effective as 1.0 mgl"1 BA in stimulating shoot proliferation. One procedure commonly used to stimulate shoot proliferation in m is decapitation (111, 111, 511, 60, 86, 96, 100). This is due to the release of axillary buds from apical dominance (”0, 96). In some instances, this procedure allows successive harvests of shoots from mother cultures (1”, 60, 86, 100). This method was also effective with 'Red Sunset' cultures. Shoot proliferation was more consistent following decapitation than when shoots were left intact. It was possible to harvest shoots twice at 10 day intervals. 67 In an attempt to stimulate or improve shoot proliferation rates, various supplements including adenine sulfate (6, 26, 39, ”1, 65) and casein hydrolysate (60, 65) are sometimes added to the culture medium. with 'Red Sunset' shoot tip cultures, addition of 100 mgl‘1 adenine sulfate to the multiplication medium, LS + 10.0 mgl'1 21p, significantly increased axillary shoot proliferation and significantly reduced the amount of basal callus. These observations were interesting since 'Red Sunset' shoots on multiplication medium developed an excessive amount of callus at the base. These results suggested that this large mass of ' basal callus may have a negative impact on shoot proliferation. Addition of casein hydrolysate to 'Red Sunset' multiplication median did not stimulate shoot proliferation although Lyrene (65) reported that this compound improved shoot proliferation rates of mm panel when used at 300 mgl”. Maximum shoot proliferation was only observed in A; rubrum 'Red Sunset' shoot tip cultures when certain conditions were optimized. These essential factors were: high levels of BA or Zip, high salt levels, adequate sucrose concentration, readily available free water, and proper temperature regime. Unfortunately, these favorable conditions also led to the development of vitreous shoots. The shoot terminals and leaves became translucent and water soaked, followed by tissue collapse and death. Guttation was normally associated with the onset of these conditions. This sequence was repeated in a cyclic pattern with the elongating axillary shoots which developed as the terminals senesced. In some instances, the entire shoot mass would succumb. Debergh et al. (27) observed similar symptoms in anara ggglymug cultures and termed this phenomenon vitrification. Anatomical studies of vitreous shoots and leaves indicate that they 68 are aquatic in nature (122) or are similar to plants growing under wet conditions (8, 62). Ziv et al. (122) noted that nianthn§.gazzgph111n§ leaves from shoots cultured in 11529 lacked cuticular waxes, had a higher water content and consisted mainly of spongy parenchyma cells. Leshem (62) also observed changes in the internal structure of vitreous carnation shoots and leaves. Two recent articles concerning plants propagated in gitng_reported on the presence of large intercellular air spaces in the mesophyll of Baby: 1dggg§ leaves (29) and Eiges,ahies needles (8). These anatomical changes and the waterlogged appearance of vitreous tissues and organs may provide clues to the mechanisms responsible for vitrification. Accanulation of ethylene is one of the first responses to the onset of waterlogging (13, 52) and ethylene is known to be the agent responsible for many of the injury symptoms associated with waterlogging (13, 51, 52) including: guttation, intanenescence formation and stem hypertrophy (”). Interestingly, each of these symptoms was observed in vitreous,AL.nuhzym 'Red Sunset' cultures. Another symptom of waterlogging is aerenchyma formation. These are large intercellular air spaces which occur in the roots and stems of aquatic plants and to some extent in other plants as an adaptation to waterlogging (52). There are two types of aerenchyma: lysigenous and schizogenous. The first type is formed by the disintegration of entire cells, while the second results from the separation of cell walls from each other (52). Kawase (52) suggested that a sequence of events lead to aerenchyma formation: first, waterlogging followed by ethylene production which stimulates increased cellulase activity and induces aerenchyma development. These structures allow plants to survive under short term waterlogging, but may not prevent plants from senescing during prolonged flooding. The 69 structures then become evidence that waterlogging has occurred (52). Although these observations are circanstantial, they suggest that ethylene accumulation in 11139 may be responsible for the development of vitreous 'Red Sunset' shoots, especially since AL ggbzgm is known to be tolerant to waterlogging (52). Recently, Kevers et al. (53) also suggested that ethylene plays a role in vitrification. They concluded that vitrification results from a burst of ethylene controlled by the peroxidase-IAA-oxidase system. To develop appropriate measures for controlling vitreous shoot formation in 'Red Sunset' shoot tip cultures, a number of factors including: agar, sucrose, cytokinin, and salt concentration; temperature regime, type culture vessel and container volume were examined. The effect which these factors had on shoot proliferation and vitrification will be considered below. The water potential of the culture medium is a critical factor in the occurrence of vitreous shoots in litgg_(27, 62, 122). It was possible to eliminate vitrification in 'Red Sunset' cultures by increasing the agar concentration to 12 gl". Other researchers have made similar observations (8, 27, 62, 122). Debergh et a1. (27) suggested that agar was responsible for the matric potential of the medium water potential and that this component was responsible for vitrification. The key to this observation was that agar does not penetrate plant tissue. Debergh et al. (27) concluded that increasing the agar concentration reduced the availability of cytokinins in the medium. What seems more plausible, however, is that raising the agar concentration creates a slight water stress (62). Such stress may reduce the growth rate of effected tissues and simultaneously reduce vitrification. This hypothesis is supported by the observation that 70 shoot proliferation rates were significantly reduced inAL zghzyn 'Red Sunset' cultures when the agar concentration was above 8 gl". Agar concentrations below 8 31'1 also reduced proliferation rates but did not reduce vitrification. This was possible due to excess free water present in the culture median. It is interesting to note that increasing the sucrose concentration in 'Red Sunset' culture median did not reduce vitrification. Debergh et al. (27) made the same observation with ana a agglymga cultures. They concluded that sucrose and mannitol effect the osmotic potential of the median water potential, but since these are tissue penetrating, they do not reduce vitrification. The response, however, may be concentration dependant. By raising the sucrose concentration to 81, Ziv et al. (122) were able to increase the number of normal Diggthg§.gazygphyllg§ shoots to 93% compared to 5”! at 31 sucrose. Sucrose concentrations below 30 gl'1 significantly reduced shoot proliferation and prevented the formation of vitreous shoots 'Red Sunset'. In culture, sucrose acts as a carbon source and when surplus carbon is available also acts as an osmotican (16). The data suggests that with 10 or 20 gl'1 sucrose there was not sufficient carbon to stimulate active growth. The role which cytokinins play in stimulating the development of vitreous shoots is a matter of speculation. Debergh et al. (27) conducted experiments with gypgganggglxmug cultures to determine if vitrification was due to the acculuation of breakdown products of synthetic cytokinins. However, after three culture periods on median lacking cytokinins, losses due to vitrification were still greater than 501. In this instance, however, a high level of vitrification was observed at all stages of development in vitrg so it would be difficult 71 to determine if the treatment was having any effect. Cytokinins did, however, play an indirect role in the formation of vitreous 'Red Sunset' shoots. Levels of BA or Zip, 5.0 or 10.0 mgl'1 respectively, which stimulated rapid growth and shoot proliferation also led to the formation of vitreous shoots. Interestingly, vitreous shoots were rarely observed at levels of BA, 21p and Kinetin which did not support active shoot proliferation. Transferring 'Red Sunset' shoots to basal medium lacking cytokinins also eliminated vitreous shoot formation. These shoots rooted and grew slowly with little or no shoot growth. 'Red Sunset' shoots were very sensitive to reductions in the levels of major and minor salts in the LS medium. However, it was not possible to determine if lowering the salt concentration had any effect on vitrification since the frequency of vitreous shoots was low in all treatments. In any event, this would not be an acceptable method to prevent vitrification because of the drastic reduction in shoot proliferation rates that occurred at lower salt concentrations. It is quite probable that the reduced growth was responsible for the lack of shoot proliferation and vitrification. It is interesting to note, however, that the first developmental response to diminish as the salt level was reduced was shoot proliferation while shoot elongation and callus growth were not significantly restricted until the salt levels were reduced to 50 and 253 respectively. It was possible to prevent vitreous shoot formation in 'Red Sunset' shoot tips cultures by holding the cultures for four weeks at 20° c followed by two weeks at 25° C. However, this was not a practical method for controlling vitrification since it prevented axillary shoot proliferation. The effect of the 200 C treatment was only temporary. This was quite apparent with cultures given the two week 200 C treatment 72 and then grown for four weeks at 25° C. Forty-three percent of the shoots harvested from these cultures were vitreous. Debergh et al. (27) also concluded that a cold treatment (”° C) was not an effective method of controlling vitrification. Their results, however, may not support this conclusion. They reported that gynang .ggglynng cultures given the cold treatment were necrotic at the end of 3-” week culture period but did not indicate the percentage of vitreous shoots. Condensation of water inside the culture vessel can lead to the development of vitreous shoots (122). Ziv et al. (122) reported that .Qignthg§.gggzgphzlln§ shoot tips cultured on a horizontal agar surface where water accumulated were more prone to injury than those cultured on an agar slant. Damage due to water condensation was also evident in 'Red Sunset' cultures. This was especially apparent in 25 x 150 mm culture tubes where shoots growing against the glass frequently became watersoaked and glassy. It was possible to reduce the frequency of vitreous shoots by placing 'Red Sunset' shoot tips in 60 ml bottles. Changing culture vessels also improved axillary shoot quality. Previously, it was implied that there was a relationship between the growth rate of 'Red Sunset' shoot tips and the degree of vitrification. Thus, reducing the growth rate may reduce vitrification. While this appeared to be the case with respect to agar, cytokinin and salt concentrations and temperature it did not seem to occur with respect to container volume. The rate of shoot proliferation and shoot elongation were reduced in 2”0 ml bottles campared to 60 ml bottles, but the nanber of vitreous shoots was higher. These seemingly contradictory results may be explained in the following manner. Shoots placed in the 2”0 ml bottles grew slower than those in the 60 ml bottles during the 73 initial four week culture period possibly due to lower relative humidity in the larger vessels. However, decapitation and transfer to fresh median stimulated shoot develoanent and excessive basal callus in the 2”0 ml bottles compared to the 60 ml bottles. It is possible that the movement of nutrients across the callus/medium interface led to the development of positive root pressure. Interestingly, guttation, a symptom of positive root pressure which occurs under conditions of high humidity (78), was frequently associated with injured 'Red Sunset' shoot tips suggesting that the large basal callus mass stimulated vitrification by allowing excess water to enter the explants. Debergh et al. (27) also studied the effects of container volane on the development of vitreous shoots and conlcuded that vitrification was not due to the presence of volatile compounds in the atmosphere. This conclusion, however, may not be warranted by their experimental design and lack of supporting evidence. First, liquid medium as used in their experiments has subsequently been shown to stimulate vitrification (53. 122). Second, they did not analyze gas samples to determine the presence of possible deleterious volatile compounds. These arguements are important since Kever et al. (53) reported that explants placed in liquid medium immediately produced more ethylene than those placed on solid medium. Control of vitrification in 'Red Sunset' shoot tip cultures was complicated by the fact that most treatments which reduced or prevented the formation of vitreous cultures also reduced shoot proliferation rates. While this situation may appear irreversible, in some cases, it has been possible to successfully control vitrification and maintain acceptable shoot proliferation by modifying culture techniques. For example, Ziv et al.(122) proposed two possible solutions for controlling 7” vitrification in Dianthng ggrzgphyllgs cultures: 1) place shoots in liquid median for a few days and then transfer to agar solidified median or 2) place shoots on agar slants and later transfer uncapped cultures to a desiccator. Both of these proposals worked on the principle that it was possible to stimulate active shoot proliferation and then control vitrification. A similar suggestion was made for flog; M cultures (8). Using the same principle, it was possible to control vitrification in decapitated 'Red Sunset' cultures by harvesting shoots before damage was observed. Culturing shoots in 60 ml bottles rather than 25 x 150 mm culture tubes also reduced the incidence of vitreous shoots. It may also be possible to reduce vitrification in 'Red Sunset' cultures by reducing the relative humidity in yitgg_as suggested by Ziv et al. (122), although placing cultures in a desiccator as they suggested seems rather impractical on a canmercial scale. Use of, a closure which would allow water vapor to escape may overcome this problem. Exogenous auxins were not required to stimulate rooting of A; .rnhrgm 'Red Sunset' shoot tips in yitgg. Proliferated shoots rooted readily in culture tubes and in 0.95 liter jars. One possible explanation for this ease of rooting is that 'Red Sunset' shoots contain a high level of endogenous auxin. The same argument was used to explain the ease of rooting of Lyggpgggigon.e§gglggtgm, 'Star Fire' (50) and ,nggallia,yi§gg§g_(11”) shoots in culture. Support for this hypothesis with 'Red Sunset' comes indirectly from two sources: 1) high levels of cytokinin were required to overcome apical dominance and 2) removing the source of endogenous auxins by decapitation allowed rapid shoot proliferation. Another explanation for the ease of rooting of 'Red Sunset' shoot tips is the phenomenon of rejuvenation. Skiskandarajah and Mullins (93) 75 suggested that culture techniques lead to physiological rejuvenation. Earlier, Welsh et al. (115) concluded that rooting of Red Maple shoots 1.1111129. was closely linked to their physiological state. Thus, conditions stimulating active growth and shoot proliferation would also enhance rooting. Changes in the physiological state of the explants in culture have been reported for a number of genera including: ,Malus (93). Rana (”1). mm (65, 66) and 11121.1 (9. 90). With some species, for example, mm (65, 66) and 111211 (9, 90), rejuventated shoots bore distinctly juvenile morphological characteristics. Lyrene (65) described two types of blueberry shoots: 1) shoots with large leaves and heavy stems resembling mature growth and 2) juvenile appearing shoots with small leaves and filamentous stems. The phenotypic juvenile shoots rooted more readily than seedling material (66). Morphological differences were also observed between 'Red Sunset' plants recovered from culture and mature, field grown trees. The leaves of the trees propagated in zitng_were generally five-lobed and cordate at the base while the leaves of the field grown trees were three-lobed and rounded at the base. Similar observations were made, however, between grafted 'Red Sunset' trees of different ages, suggesting that these differences in leaf shape may only reflect the growth rate of the trees. The ease of rooting of 'Red Sunset' shoots has another implication. An essential feature of an economically feasible micropropagation system is the ability to root a high percentage of shoots enmasse over a relatively short period of time. This has been demonstrated with 'Red Sunset'. It may also be possible to root 'Red Sunset' shoots under septic conditions in a high humidity chamber. This approach has been used successfully with Kalmia (63) and fistula (69) microcuttings. 76 The uniformity of 'Red Sunset' plants propagated in 11129 is an indication that this cultivar is stable genetically. This means that 'Red Sunset' cultures can be maintained for long periods of time without needing to start new cultures on an annual basis and go through the initial problems involved. It also indicates that the axillary shoot proliferation system employed here is a relatively safe method for propagating plants in yitgg, Others (”1, 63, 67, 69, 76) have made the same observation. SUMMARY Aseptic, actively growing cultures of Age; gghggn 'Red Sunset' were established using a preconditioning treatment which consisted of transferring shoot tip or node segment explants, 3-” cm , at 3-” day intervals to fresh LS basal median or with 1.0 mgl"1 BA or Zip. This procedure made it possible to select actively growing explants and to discard contaminated and phenol damaged tissues. Holding surface sterilized 'Red Sunset' explants in a humid environment prior to dissection also helped to reduce injury by polyphenols. The most effective cytokinin in stimulating axillary shoot proliferation in 'Red Sunset' shoot tip cultures was BA with 5.9 shoots/explant at 5.0 mgl"1 followed by Zip with 3.0 shoots/explant at 10.0 mgl'1. It was suggested that the high level of cytokinin required to stimulate maximum shoot proliferation was possibly due to high levels of endogenous auxin in the 'Red Sunset' shoots in vitro. This hypothesis was partially supported by the observation that shoot proliferation was most consistent following decapitation. Axillary shoot proliferation was also stimulated by the incorporation of 100 mgl'1 adenine sulfate in the LS + 10.0 mgl-1 21p multiplication median and this concentration also significantly reduced basal callus fresh weight. These observations were interesting because they implied that excess basal callus had a negative effect on shoot proliferation. Conditions in litre which stimulated optimum shoot proliferation in 77 78 'Red Sunset' cultures including: .high levels of BA or Zip, high salt levels, adequate sucrose concentration, readily available free water and proper temperature regime also led to the formation of vitreous shoots. It was possible to control vitrification in decapitated 'Red Sunset' cultures by harvesting shoots before damage was observed. Culturing shoots in 60 ml bottles rather than 25 x 150 mm culture tubes also reduced the incidence of vitreous shoots. No exogenous auxins were required to stimulate rooting of 'Red Sunset' shoots. g yitzg. This observation agrees with the hypothesis proposed earlier that 'Red Sunset' shoots contain a high level of endogenous auxin(s). The ease of rooting of 'Red Sunset' shoots i3 ,yitzg could also be explained by the phenomenon of rejuvenation. With few exceptions, plants recovered from culture were uniform phenotypically. This was an indication that 'Red Sunset' is genetically stable and that this cultivar can be safely propagated in yitgg using an axillary shoot proliferation system. 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