FACTORS AFFECTING FLOWERING IN LETTUCE By Lawrence Rappaport AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1956 Approved ABSTRACT Seed vernalization, plant growing temperatures, daylength and chemical growth regulators, interacting, development markedly influenced flowering and seedstalk in Great Lakes head lettuce. of a "physiological separately and index" of flowering bers preceding a distinct developmental The superiority such as leaf num­ stage, in contrast to a growth measurement such as stem elongation, was demon­ strated. Photoperiod and air (but not soil) temperatures were shown to developing influence the number of leaves preceding the inflorescence. Flowering was accelerated in plants grown from moist seed germinated at 60 to 70°F for 24 to 48 hours and subjected to 40°F for a minimum of 13 days or more. Seed germinated 3 days at 60 to 70°F were not effectively vernalized at 40°F for 16 days. Periodic temperature interruptions at 60 or 90°F for two hours daily or at three day intervals did not nullify the vernalizing effect of cold exposure. Plants from lettuce seed which had been soaked at 60 to 70°F and air dried 6 or 12 hours prior to chilling at 32 or 40°F were delayed pared with those from vernalized seed. in flowering as com­ When Maleic Hydrazide (20 or 40 ppm), was applied during vernalization, plants pro­ duced visible flower parts at a lower node than those from seed vernalized in water alone. Growing lettuce plants at night temperatures above 65°F subsequent to vernalization accel­ erated seedstalk development without preceding head formation. Below 65°F vernalized plants first produced a high percent­ age of firm, vegetative heads and then flowered. Non­ vernalized plants flowered only at night temperatures above 65°F. Earlier flowering resulted in plants exposed contin­ uously to warm temperatures as compared to alternating cool (50 F) and warm (70°F) night temperatures. potential for flowering Apparently, the is greater following seed vernal­ ization, but expression is subsequently delayed by continuous o night temperatures of 50 F. Soil temperatures influenced the rate of seedstalk development, stimulus per se. but not the vernalization At high soil temperatures (64 and 70°F) flowering (lays to anthesis)was promoted, while at low temper­ atures (50 and 57°F) flowering was delayed. A decrease in percent seedstalks occurred as soil temperatures declined from 71 to 50°F. Photoperiod markedly affected flowering in vernalized head lettuce. The minimum night temperature following vernalization which favored early flowering was reduced from 65 to 60°F when plants were grown at a daylength of 16 hours. At 60°F with a nine-hour day, and non-vernalized plants formed firm, flowered simultaneously, both vernalized vegetative heads which thus the effects of seed vernaliza­ tion were nullified by a short daylength. The delaying effect of cool temperatures (both soil and air) and relatively short days on seedstalk development demonstrated greenhouse, was verified in field studies. in the early Spring formed heads in the Plants grown irrespective of previous seed vernalization and treatment with chemical growth stances. Similarly, endive (variety, Full Heart Batavian) displayed marked sensitivity to cool tures during early growth. sub­ (below 60°F) tempera­ Overwintered plants and seedlings, plants from seed sown out of doors April 1, or transplanted to the field May 21, after vernalization flowered earlier than those from seed sown after May 1 or from non-vernalized transplants. Possible biochemical differences induced in lettuce seed by vernalization were studied by subjecting the ether extracts from vernalized and non-vernalized seedlings to paper partition chromatography. Fluorescent spots detected with ultra-violet light and occasional colored spots de­ tected with Salkowski and Ehrlich*s reagents were thus isolated. R^ values possibly comparable to those of 3-Indole- acetic Acid, Tryptophan, Indoleacetonitrile, and Ethyl Ester of Indoleacetic Acid developed both with water and Isopropanolammonia water-water (8:1:1 V/V) appeared on chromatograms of extracts of both vernalized and non-vernalized seedlings. consistent differences were evident. No Extracts developed with the 8 : 1:1 solution on paper yielded spots which fluoresced in ultra-violet light at 0.52, an value at which no bio­ logically active indole compound has been identified insofar as the author is aware. Bioassays for L-tryptophan revealed that .00127 and .00293 per cent occurred non-vernalized seedling extracts, value of L-tryptophan in vernalized and respectively. in vernalized seedlings The low suggests direct utilization of L-tryptophan or conversion during cold expo­ sure to another Acid. indole compound, conceivably 3 - Indoleacetic FACTORS AFFECTING FLOWERING IN LETTUCE By Lawrence Rappaport A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1956 Approved ProQuest Number: 10008686 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008686 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ACKNOWLEDGEMENT The author is deeply indebted to Dr. S. H. Wittwer whose friendliness, guidance and criticism during the performance of the research and writing of this manuscript are genuinely appreciated. Dr. H. N. Fukui is acknowledged for his suggestions and aid in the performance of the biochemical studies. The interest and technical assistance of Drs. S. K. Ries, and D. H. Dewey, Lastly, R. are gratefully acknowledged. appreciation is expressed to Drs. L. Carolus, S. T. Dexter, guidance committee, L. M. Turk, and H. M. Sell, members of the and to Dr. H. B. Tukey for their sugges­ tions on the graduate program and the manuscript. TABLE OF CONTENTS PAGE INTRODUCTION 1 REVIEW OF LITERATURE 2 Foreword The Effect of Temperature on Flowering The Effect of Daylength onFlowering Flowering and Chemical Growth Regulators Biochemical Studies Related to Flowering THE PROBLEMS FOR INVESTIGATION GENERAL PROCEDURES Vernalizing Seed GrowingPlants Control of Temperature Control of Daylength Measurements of Flowering Responses Statistical Methods EXPERIMENTAL I. FACTORS 2 3 6 10 12 14 15 15 15 16 16 16 17 19 INFLUENCING VERNALIZATION Duration of Chilling Age of the Plant Temperature Moisture Light Quality and Duration Chemical Growth Regulators II. FLOWERING AS AFFECTED BY THE PLANT GROWING ENVIRONMENT SUBSEQUENT TO VERNALIZATION Night Temperature 1954 Tests 1955 Tests Soil Temperature Photoperiod 1954 Tests 1955 Tests Field Experiments 19 19 19 22 24 26 27 31 31 31 33 38 38 43 43 46 page m. BIOCHEMICAL ASPECTS OF LETTUCE SEED VERNALIZATION 51 First Seed Lot Vernalizing Seed Extraction of Fresh Seedlings Extraction of Frozen Seedlings Chromatographic Techniques 51 51 51 52 53 Second Seed Lot 55 r c Vernalizing Seed Extraction of Fro-zeji Seedlings Determination of L-tryptophan Vernalizing Seed Extraction and Determination of Free L-tryptophan DISCUSSION ENVIRONMENTAL FACTORS AFFECTING FLOWERING OF LETTUCE Indices of Flowering Night Temperature Photoperiod Soil Temperature THE VERNALIZATION PROCESS Duration of Chilling Age of the Plant Temperature Patterns During Vernalization Moisture During Vernalization Light Quality and Duration Chemical Growth Regulators ^ 55 6U 60 60 62 62 52 54 5o 59 69 69 7* 72 7 j; ^ BIOCHEMICAL STUDIES 76 SUMMARY AND CONCLUSIONS 70 LITERATURE CITED C2 LIST OF FIGURES PAGE An Immature Inflorescence 13 The Effects of Night Temperature on Seedstalk Devel opment in Great Lakes Lettuce. Left to Right: 70, 6 60, 50°F. Above: Vernalized. Below: Not Vernalized 34 The Appearance of the Soil Temperature Experiment Showing the Comparative Differences in Development of Great Lakes Lettuce Growing in the Various Tem­ perature Tanks (A - 71 F; B - 64 F ; C - 57°F; D - 50°F) 39 The Influence of Soil Temperature on Days to Primordia and Anthesis, and Per Cent Seedstalks of Vernalized Great Lakes Lettuce Grown at 70°F Night Air Temperature 41 The Effects of Temperature and Photoperiod on Seed­ stalk Development in Great Lakes Lettuce: A - 70°F t 16-Hours; B - 70°F, 9-IIours; C - 60°F, 16-Hours; D - 60°F, 9-Hours. Above: Vernalized. Below: Not 43 Vernalized Interaction of Daylength (9 and 16-Hours) and Temperature (60 and 70°F) on the Number df Days to Anthesis (Vertical Axes). Shaded Area Indicates Differences Resulting From Seed Vernalization 45 Effect of Vernalization on Flowering in Endive (Variety Full Heart Batavian). (Left) A Flowering Coldframe-grown Plant, Seeded December 29, 1953. (Right) A Vegetative Plant, Started in the Green­ house April 12, 1954. Photographed August 11, 1954 40 LIST OF TABLES PAGE I. II. The Duration of Vernalization Necessary for Acceleration of Flowering in Great Lakes Head Lettuce Effect of Age of the Plant Prior to Vernalization on Subsequent Flowering of Great Lakes Lettuce 20 21 i III. IV. V. VI. VII, VIII. IX . X. XI. XII. Flowering of Great Lakes Head Lettuce as Affected by Interruptions of Vernalization by 2-Hour Exposures to 60 or 90°F Temperatures Daily and at 3-Day Inter­ vals. 23 The Influence of Moisture Before and During Seed Vernalization on Subsequent Flowering of Great Lakes Lettuce 25 Flowering^of Great Lakes Head Lettuce as Affected By N-meta-Tolylphthalamic Acid (7R5), Maleic Hydrazide (MH)# and 2,3,5-Triiodobenzoic Acid (TIBA) Applied During Seed Vernalization 29 The Effects of Night Temperature on Growth, Flowering, and Seed Maturity of Vernalized and Non-vernalized Great Lakes Lettuce (1954) 32 Flowering of Great Lakes Lettuce as Influenced by Night Temperatures Following Vernalization (1955) 35 Flowering of Great Lakes Lettuce as Affected by 50 and 70°F Night Temperatures Following Ver­ nalization. 37 Effect of Soil Temperatures on Flowering of Ver­ nalized Great Lakes Lettuce Grown at an Air Temperature of 70°F 40 The Effect of Photoperiod, Night Temperature, and Vernalization on the Flowering of Great Lakes Lettuce 44 Rf Values of Possible Naturally Occurring Indole Compounds Separated by Paper Chromatography from Ether Extracts of Vernalized and Non-vernalized Great Lakes Lettuce Seedlings (Detection by Ultra-violet Light) 56 R£ Values of Possible Naturally Occurring Indole Compounds Separated by Paper Chromatography from Ether Extracts of Vernalized and Non-verna1 ized Great Lakes Lettuce Seedlings (Detection by Ultra-violet Light) 59 1 INTRODUCTION Seed production from non-bolting varieties of lettuce ofterv necessitates slashing the mature heads to permit emergence of. vigorous seedstalks, is costly, a practice which encourages disease transmission, ineffective (18). and may be A method to circumvent this operation would materially reduce production costs. Vernalization (chilling partially germinated seed) as a technique for promoting flowering and seed production has been employed successfully in a variety of important crops (1, 15, 22, and 81). 23, 24, 25, 32, 33, 34, 35, 50, 72, 73, In lettuce, however, conflicting reports occur regarding the effectiveness of vernalization in accelerating seedstalk development without prior head formation 23, 42, (1 , 24, 45 and 70). Since vernalization may hold promise in seed pro­ duction these apparent inconsistencies suggested a more critical evaluation of the internal and external conditions favoring flowering and seedstalk development in lettuce. Such a study might add not only to our knowledge of flower formation in head lettuce but in other crops as well. 2 REVIEW OF LITERATURE Fore w o r d i For centuries man has sought to control the growth and reproductive development of cultivated crops. However* it was not until Sachs published his classic treatise (65) tnat the scientific approach ology was adequately presented. different By demonstrating that species possess minimal* temperature requirements* investigations in plant physi­ optimal and maximal he provided the basis for later on the temperature requirements, cific responses such as flowering for spe­ in many crops. Perhaps without realizing the enormity of his dis­ coveries in “ electro-horticulture" Bailey (4) described the response to day length of "spinage" which "ran to seed" when exposed to the electric arc light. Allard Later Garner and (21) discovered photoperiodism which has since been a source of intense scientific interest. Coincident with the study of daylength considerable attention has been focused on the intensity and quality of light in relation to physiological responses in plants (5,6*16). Investi­ gators are now making a concentrated search for the specific portion of the spectrum and the pigments ated with associ­ such phenomena as related to photoperiodic flower induction and vegetative development (38). 3 The suggestion by Sachs (55) that transmissible f 1 ower-forraing substances occur in plants, quent and the subse­ isolation of natural and synthetic compounds exhibit­ ing biological activity, (3, 29, 30, 44, 50, 63, 77) have provided a thread which may eventually bind together the many factors responsible for vegetative and reproductive expression. Because flowering in many plants mately related with temperature, growth regulators, comprehensive day length, is so inti­ and chemical consideration of these factors study of flowering in a is imperative. The Effect of Temperature on Flowering: The acceler­ ation of flowering by chilling or freezing wheat seed was reported by Klippart (32) in 1857: "To convert winter into spring wheat nothing more was necessary than that the winter wheat should be allowed to germinate slightly in the Fall or Winter but kept from vegetation by a low temperature or freez­ ing until it can be sown in the Spring." Gassner (22) in 1918 found that when seed of cereal crops were planted on progressive dates, near-freezing, those exposed to as compared to higher temperatures, flowered earlier and developed most uniformly. The publication by Lysenko (39,43) on the promotion of flowering by vernalization renewed interest in tempera­ ture studies among investigators who, for a time, had been diverted by the newly discovered phenomenon of photoperiodism. A classic series of papers by Purvis and Gregory 4 (23, 25, 26, 27, 47, 48, 49, 50) and their associates in England have helped to elucidate the factors related to vernalization conceptions (39, 43). in cereals and to dispel certain of the mis­ introduced by Lysenko and his colleagues Principally^they found that the stimulus which is perceived by the embryo meristem rye (25, 26) and, likely, (48) causes acceleration of flowering (47). concept of "minimal McKinney and Sando (34), influenced (23, 47). The leaf number" preceding the inflores­ cence is characteristic of the work of Purvis (81), Lang in winter The response of vernalized grain was by subsequent temperature and photoperiod the apical (40), Leopold (47). (35, 36), Wittwer et a 1 and recently Gott (23), have re-empha­ sized the reliability of leaf numbers as a specific physio­ logical index of flower expression. In the United States, McKinney and Sando (40) working with cereal crops found results comparable to those of Purvis and Gregory, but Sprague (62) showed that "iarovization" caused very little acceleration of flowering in corn. Sen and Chakravarti (57) reported that only a mini­ mum of pregermination was necessary to accelerate flowering in chilled mustard seed. initiate germination, Seedlings soaked long enough to but not to permit emergence of the 5 radicle, without could be vernalized and then dried six years loss of the stimulus. Melchers (41) found that Hvoscvamous nicier was in­ duced to flower without customary cold treatment when grafted to a flowering plant. Thompson and coworkers (67) investigated the response to cold temperatures of a series of horticultural crops. In experiments by Knott, et^ aj, (33) lettuce seedlings were exposed at various stages of development to a temperature of 40°F and -5°F. Varying with variety vernalization at 40°F for 10-20 days accelerated flowering, while in results comparable to those of Rudorf and Stelzner (54), freezing temperatures retarded germination and did not influence flowering. In contrast to seedlings not vernalized, seed­ lings grown at 70 to 80°F or at 50 to 60°F produced seedstalks earlier. At 60 to 70°F vernalized plants produced loose heads. Reimers (79) in 1938 found a difference response of lettuce to vernalization. only germinating seedlings, in varietal He concluded that as compared to those with the first true leaf expanded, were receptive to cold exposure of 2.5 to 5°C and produced plants accelerated. In contrast, Warne in which flowering was (76) reported that lettuce seedlings germinated three days were not effectively ver­ nalized. He found that compared with non-verna 1 ized see T j* * h O CO — 1 H 1—1 0 >rr •—1 • LO O O • CM X 1 ■ O ' CM r-H I-H • X 1 S J it e ^ s p a a s LO O ' • LO •-4 CM • X* NO M LO • CO CM M O' • i-H LO i-H o m CO /-N O . -Q f"" QI w > 1 < vO *“H LO ♦ • • O ' i-h 0 CM CM CO r— i rH CO LO w O O • CO CM • sO O <—( E-» Temperature for remainder of the growing period L.S.D. may be used to compare any pair of measurements %H o (0 (b) (c) THE EFFECTS OF NIGHT TEMPERATURE ON GROWTH, FLOWERING, AND SEED MATURITY OF VERNALIZED AND NON-VERNALIZED GREAT LAKES LETTUCE (1954) t '* to -o 0 0> u. N ° CO •H W o +, h ^ . h O CO _ 2 : *3 E x a w o ^ * i“H • nO -Q LO S-" vO ia XS /- v o > t - c o O ' i-H vO • • • H H O CO CM i-H O O • CO CM » O ' O •-H ■O • X 1 CO I-H X* • LO LO ■—1 •iH CO • —» CO vO • I 'CM O * t 'O (H CO • X CM —) CM • i-H a 10 H o> i-H > CO 0 • O ' X 1 • LO CO i-H O * X 1 CO CO N CO u O lf lH • * * CM O h O LO sO 0 r*H • l'IO i-H CO CO CO tj o O LO s | s > ix e x s p o a s o m o CM CD -O a> -Q W CO 3 •H 09 M CO CO CD > S *G X 1 X< O LO LO •<*4 O ' O ' 4-> i-H i-H CO o» * G i-4 O ' O CO CM 4-> 1—f tA cd i s h a> 0 > ^ H M W H CO CL CO CO 33 +-> n XJ CD O cn 0 9 Q> C w • 1-4 G Oh O 0 I-H &> > 0) •O ' (A •M 0 CO CD M O 1—< CM O G •M -M CD »—( JO H (A -H > V) 9i W +-> M CO CL CA CO (1) O +-> 5-1 a> CA 3 >» 0 CO M -3 Qlt. -G 4-> C C« >» 4-* •iH M 3 +-> CO E X5 * CO 0 O ■M (A >» CO a 33 the number of days preceding the visible flower parts. vernalized plants, however, three weeks later. hastened anthesis Non­ produced visible flower parts A 70° (as compared to a 6 5 ° 0 temperature in vernalized plants by 11.1 days. parison to those not vernalized, In com­ seed maturity of vernalized plants was hastened. The effects of differential temperatures at progressive developmental stages on flowering of lettuce were determined by growing plants immediatly after vernalization at night temperatures of 50 and 65°F and shifting after 20 o days to 65 and 70 F, respectively. Flowering jected to these temperature patterns, in plants sub­ as compared to those grown continuously at 65 and 70°F, was not influenced sign if icantly. 1955 T e s t s : In a more detailed study initiated December 1954 plants from vernalized and non-verna1ized seedlings were grown at night temperatures of 50, 60, 65, and 70°F. Vernalized plants produced no seedstalks when o o grown at 50 F, 25 per cent seedstalks at 60 F and at 65 and 70°F 100 per cent seedstalks without prior head formation (Table V I L and Figure 2). No seedstalks nor flowers de­ veloped from non-verna1 ized plants maintained at 50 or 60°F. As in 1954 (Table VI) fewer leaves preceded the appearance of the developing inflorescence in vernalized plants as com­ pared to those not vernalized. Leaf numbers in vernalized 34 Figure 2, The Effects of Night Temperature on Seedstalk De ­ velopment in Great Lakes Lettuce, Left to Right: 70, 65, 60, 50°F, Above: Vernalized, Below: Not Vernalized 35 • i—i CO . CO nO o i-H l-H CO CO r-H o o rH LO r—1 l-H O ' CO rH o o • O ' a . CO (JO uJ 0 CO • CM -- o o^> LO r '• r CM FLOWERING OF GREAT LAKES LETTUCE AS INFLUENCED BY TEMPERATURE FOLLOWING VERNALIZATION (1955) NIGHT Lb O o CO • H CO Uh 'O © N •H rH CO c O in o . , vs 4-> C © E bn o LO CO o vO jh © > o CO l-H LQ CM vO r-H 1A O CO o • fi o rH LO a © os U-. o G u n s mo TI © JH 3 VS A © E o OW O f-t •iH © CL, TT LO rr Lb o o hxs © N •iH l-H CO c w © > o Vh © o CO i-H lO •o rH CO CO • Lb © LO vO © > < r— TT ' o CO *—1 © JH © Cu t^ nO i—1 © 4-> 1 1 1 1 l" CO rH o o w o X> © vs 3 z Lb buiaaMOTT 05 ss •IH Cu o i-H © © i-H XJ CO •iH TO © JH 3 VS jh © CO © >> E o © i-H Lb O o NO O O LO c A © a o 3 O © *rH ■ 3 4-» 4-5 A •rH E C © o VS © > JH © o T 3 i-H «*H o e +-» -iH VS © > © © l-J © i-H -O vs •iH +-> vs M ■iH © > cu vs ■rH vs © -C 4-> © © O 4-5 O 4-> JH © 5 vs O i— H © <+H Q VS >» © a 5 M © -SC •-H © 4-5 VS XJ © © CO o > © E a • CO * —1 © -C E-h /-N © '—' 36 plants decreased ture. significantly with each 5°F rise in tempera­ Plants grown at a 65°F night temperature subsequent to vernalization, were, ment. although partially covered with black cloth nevertheless, exposed to light from an adjacent experi­ It is interesting, similar treatments therefore, (Table VI) that compared with in the 1954 experiment, leaf numbers preceding the visible flower parts and days to anthesis were reduced. At 70°F vernalized plants produced 3.6 fewer leaves than those at 65°F. as compared to 65°F The effect of 60 in delaying flowering despite previous vernalization was indicated by the greater number of leaves and days preceding the appearance of visible flower parts and Plants not vernalized and grown at 65°F o and those vernalized and grown at 60 F flowered simultaneously days to anthesis. and approximately the same percentage of plants produced seed­ stalks without prior head formation. The influence of cool temperatures suppressing and warm temperatures promoting flowering following vernalization of Great Lakes lettuce seed was further studied by growing some vernalized plants continuously at 50 or 70°F night tempo eratures while others were maintained at either 50 or 70 F for 66 days and then interchanged. Plants grown continu­ ously at 50°F were strongly vegetative producing heads despite previous vernalization, duced while at 70°F vernalized plants pro­ 100 per cent seedstalks. However, only six days 37 Q LO *I _} ' vO o CO CO 230 'w'O LO I r- CO CO COO wh, FLOWERING OF GREAT LAKES LETTUCE AS AFFECTED BY 50 AND 70°F NIGHT TEMPERATURES FOLLOWING VERNALIZATIO /->0 JQO CO . 73 w N 01 I N r-^O •rH C OLO i“ 1 r /»-N CO 00 fi CD M tS4 H-> 02 O CO > O O h- — I i-H a O O 01 05 "O' Vh —* O 23 O o • r-vLO Oj at i > ■—'o < "O f'-w 6) CM r— « CM h- V) H-> S3 0> E G> u 3 s om LO N J O •rH w O r-H I'- LO co c/1 CO 01 E <*H O s om OJ CL. LO CO CO COr-% | B CO C (50 J-4W UO o 1*4 2> o o o 01 w CO O' LO CL. 2 CO C5> O 2 co b *rH u- o o s^jeaspaas LO om JH •rH b oi *•4 01 o 73 r—H C+H O E CO CO CO • s CO O O > S-l Cl VH o C O •rH vO i-H cn 02 4-> •rH CO <3 > co CO k-l 3 <4H +■> 0> o o 23 W CO 3 o 73 01 +-> 5 vO O HO CO w b CL, <0 a> *o i-H (1) o 02 01 W W 02 rH 23 C O 22 3 3 CO 4-> -»-> E •rH +■> co co CO •rH W M O (!) Q > w co CL, o w +-> ai * Cu Cu * S E CO o gi • E-h H .-I s CO o >-> r-H CO a co S i -x- 38 separated the appearance of the developing inflorescence of vernalized plants grown continuously at 70°F and those held for 66 days at 50°F and then shifted to 70°F (Table VIII). Flowering was greatly delayed when high preceded low night temperature following vernalization of the seed. Soil Tempe rat ure : The influence of soil temperature on flowering of lettuce was studied by growing vernalized (40°F for 20 days) and non-vernalized plants in 2-gallon glazed crocks of soil in controlled temperature tanks maintained at 50, 57, 64, and 71°F (Figure 3). (52) Thus vari­ able temperatures were provided within the same greenhouse air temperature of 70°F. Only at the highest temperature did non-vernalized plants produce seedstalks without prior head formation. Although ing inflorescence leaf numbers preceding the develop­ (primordia) of vernalized plants were not affected significantly by soil temperatures, the occurrence of this stage and anthesis of the first flower were accel­ erated above and delayed below a soil temperature of 64°F. The percent seedstalks produced without prior head formation from vernalized plants decreased as soil temperatures de­ clined from 71 to 50°F Photoperiod: of photoperiod, (Table IX and Figure 4). The separate and interacting effects night temperature and vernalization on flower­ ing of head lettuce were evaluated in a series of experiments. 39 tn DxC a 1 CO •rt H O CD -4 O J 3 > • pH +-> H-> CO CO M U CD CO Q . o, E •pH 5 O o J3 J3 +-> CO 3 +-> • p H b CD 3 o E •fH •rt M s CD o CU M X o Cxi CD 0) O W 3 3 +-> +-> +-> CO CD . w t-J »—> cd U- C l. w o E «M LQ O 1 Sh O CD o s CO W CO 0> o. H-> 3 O 0> E •o . u< o o r - t TT CD vO > 0) o*Q < | 03 3 CD • p H . -3 U« WO H CD i“H o « 3 CO O 1 u CD CD < H <«-H 3 CD . fifk A 40 TABLE IX EFFECT OF SOIL TEMPERATURE ON FLOWERING OF VERNALIZED GREAT LAKES LETTUCE GROWN AT AN AIR TEMPERATURE OF 70°F Soil Temperature 71 °F 64°F 57°F 50°F (Ave. of 6 Replicates) L.S.D. No Significance Leaves to developing inf 1orescence 32 33 34 31 Days to visible flower parts 89 93 109 116 6.4 Days to first anth e s is 108 112 135 146 13.2 seedstalks without prior heading 100 83 33 33 % 1% 95 __ percent seedstalks 41 150 + 130 120 days toprimordia days to anthesis 140 II0-. 105 90 50 57 64 71 ROOT T E M P E R A T U R E * F Figure 4. The Influence of Soil Temperature on Days to Promordia and Anthesis, and Per Cent Seedstalks of Vernalized Great Lakes Lettuce Grown at 70°F Night Air Temperature 42 1954 Tests: seedlings were Beginning December 29, 1953 lettuce exposed to 0, 7, and 24 hours of light during vernalization. germinated 24 hours, On January 18, with seedlings they were transferred to a 50°F greenhouse where half the plants were placed under contin­ uous lighting (10 foot-candles from mazda lamps), and half under normal daylength. It was observed that vernalized plants, to those not vernalized, as contrasted irrespective of previous light condi­ tions produced a high percentage of seedstalks without prior heading when grown with a continuous light. In contrast, both vernalized and non-verna1ized plants grown under natural day-length produced heads. 19 55 T e s t s : Vernalized and non-vernalized seedlings were shifted to two night temperatures two photoperiods (60 and 70°F) and (9 and 16-hours). The developmental responses are shown in Table X and Figure 5. In non-verna1 ized plants seedstalk development occurred only under a 16-hour day and a 70°F night tempera­ ture. With all other photoperiod and temperature combinations plants not vernalized produced firm vegetative heads. Flower­ ing of vernalized lettuce was markedly promoted by a 16-hour day and 70°F temperature. No seedstalks developed without prior head formation when lettuce, grown at a 9-hour photoperiod. vernalized or not, was Figure 5, The Effects of Temperature and Photoperiod on Seedstalk Development in Great Lakes Lettuce: A - 70°F, 16Hours; B - 70°F# 9-Hours; C - 60°F, 16-Hours; D - 60 F, 9-Hours, Above: Vernalized. Below: Not Vernalized ♦ a CO • l—i * s CO '6 ^ CO • <—t U o • CM CO • (—1 1—I | CO CO i—i MD CM H r-i sO i—1 U 3 o THE > 2 1 o S3 O CM CO > O o I—I ° j /—N CO a> to U a > Z o | m « O CO CO *—i o f- o S3 J Ci O CO r—4 V <4-1 o CO u a © > •S3 > < vO r—1 CO vO 1—1 rrH CM iH pH LO CO i—1 o o o 1 | CO hi—I vO o s O i—l © CO B O H a> 1 1 o 4-» 3 o -o © © CO +-» 3 CO O O o o *—i CO © © > © o r— 1 0> *t3 •— l -Q W CO =3 •I-I CO H CO CO S +-> B a) E © w 3 *0 co © CO N •r-i | CO S A O 2 >* B *0 CO B CO © H TJ co © rH * •o LO CO EFFECT OF PHOTOPERIOD, ON THE FLOWERING NIGHT TEMPERATURE, AND VERNALIZATION OF GREAT LAKES LETTUCE co 9 B 4r» •H > CO 0} -J S3 CP 4-> B •1-1 *1-1 5 -a CO CO •H CO > o. o (O © H M +J © S CO O >-»I—I ** CO M •* J3 •1-1 CO CO © +-> -C O 4-» CO CO S >» CO CO D Q i—i CO w 4-> O CO *fH X3 M a> &■ 0) co CL N B >>•*■< 3 E > Z # Q T3 • B CO CO • •— I > /-N CO 45 ^ ^ N o t vernalized 16 hrs. Days 9 hrs. to anthesis Vernalized 9 hrs. 16 hrs. Day l ength Fiaure 6. Interaction of Daylength (9 and 16-Hours) and Temperature (60 and 70°F) on the Number of Days to Anthesis (Vertical Axes). Shaded Area Indicate Dif­ ferences Resulting from Seed Vernalization 46 Leaves produced prior to flowering of vernalized plants grown at 60 and 70°F with long days varied only slightly. However, as compared with at 70°F and a long day, vernalized plants, those not vernalized, produced 26 fewer leaves preceding the developing inflorescence. The combined effects of long days and high temperatures in promoting flowering were further indicated by the fewer numbers of days preceding visible flower parts and anthesis of the first flower in vernalized as compared to non-vernalized plants. Interestingly, vernalization also accelerated flowering at 60°F without previous head formation under a 16-hour photo­ period, thus nullifying the effect of the cool temperature in preventing seedstalk development. with the results of Andrew (1). This is in accordance Irrespective of temperature vernalized plants and those not vernalized, when grown at a 9-hour photoperiod, flowered (Table X). initially formed firm heads and then The number of days preceding anthesis, however, were not significantly different. A 16-hour day especially favored flowering of vernalized lettuce in that all plants produced seedstalks without prior head formation both at 60 and 70°F although the 60°F temperature delayed flowering and seedstalk development. Figure 6 shows dia- gramatically the interaction of temperature, photoperiod and vernalization on flowering of Great Lake lettuce. Field Experiments: To determine whether seed over- 47 wintered outdoors in Michigan is effectively vernalized and induced to flower without preceding head formation, and to evaluate the effects of outdoor temperatures on flowering of vernalized salad crops, designed. Rapids a series of field experiments were In December 1953, lettuce, seed of Great Lakes and Grand and Full Heart Batavian endive were placed in three-inch pots of soil sunk in a coldframe groundbed. The developing seedlings were transplanted to a field plot May 29, 1954. Other seedlings not exposed to natural out­ door cold, were started May 8 in a 60° greenhouse and on June 6 were transplanted to a field plot. Great Lakes lettuce grown from over-wintered trans­ plants formed loose heads and flowered about two weeks earlier than greenhouse-grown plants. Grand Rapids leaf lettuce was not affected by over-wintering the seed in the coldframe. However, a remarkable sensitivity to cold- exposure was exhibited by endive. Overwintered plants produced seedstalks directly and flowered two months earlier than greenhouse-grown transplants which first produced a characteristic rosette, (Figure 7). Thermograph records indicated that night temperatures remained below 60°F during the heading period. To further evaluate the effects on flowering of over­ wintering seed and seedlings of salad crops, a field plot was designed on August 1, 1954 as a randomized block consist- Fiqure 7. (Left) tative Effect of Vernalization on Flowering in Endive (Variety Full Heart Batavian). A Flowering Coldframe-grown Plant, Seeded December 29, 1953, (Right) A Vege­ Plant, Started in the Greenhouse April 12, 1954. Photographed August 11, 1954 48 49 ing of three replicates. Four rows of oats were seeded August 5 between each of the plots for the purpose of holding the winter snow cover. Eight salad crop varieties were seeded monthly in each plot from August 1 to December 1, 1954, and on April 2, 1955. For control comparisons seed­ lings of the same crops were vernalized and grown in the usual manner in a 60°F greenhouse to the three-leaf they were shifted to a cold-frame (May 14). stage, when A week later they were transplanted to the field simultaneously with non­ vernalized plants. The following varieties with their corresponding stock numbers and sources were utilized in the exp erim ent : Variety Stock Number Source Great Lakes 11447 Cornell 456 15329 do Bibb 15490 do Grand Rapids 15057 do Pennlake 15043 do Full Heart Batavian (Endive) 15540 do No. 352-3-2 Dessert Seed Company, El Centro, California 535504 Rohnert Seed Company, Gilroy, California 407 Great Lakes Without exception, Ferry Morse Seed Com­ pany, Detroit, Michigan irrespective of planting date or ver- nalization under controlled conditions (40°F for 20 days), the head lettuce varieties produced firm marketable heads and later weak seedstalks. Flowering in the leaf lettuce varieties was not significantly accelerated by overwinter­ ing or vernalization at 40°F. those of the previous year overwintered seed, vernalized at 40°F, contrast, In results comparable to (Figure 7) endive plants from from seed sown April 2, or from seedlings flowered from June 27 to July 10. In non-vernalized transplants or seed sown after May 1 in the field produced large vegetative heads and did not flower until September 1. Records showed that average night temperatures remained below 60°F period. during the heading 51 III. BIOCHEMICAL ASPECTS OF LETTUCE SEED VERNALIZATION The purpose of this study was the possible isola­ tion and characterization of indole compounds exhibiting biological activity from vernalized and non-verna1ized seedlings. Nitsch (44) Essentially the same techniques as described by for the isolation of free auxin from strawber­ ries were utilized. First Seed Lot: Vernalizing S e e d : Several one hundred gram lots of lettuce seed were spread evenly over moistened filter paper placed in plastic serving trays on May 13, 1955. tional layer of wet filter paper was spread over the An addi­ seed and the covered trays of seed were exposed to 60°F for 48 o hours prior to storage at 40 F for 20 days. On May 29 addi­ tional trays of seed were prepared Four days later half the seed were and maintained at 60°F. frozen at -5°C. Extraction of F resh S eedlinas: The fresh seedlings were extracted immediately by grinding with sand in the pre­ sence of peroxide-free ether for two to four hours in the o dark in a water bath maintained at 0 to 10 C. Extracts of the vernalized and non-vernalized seedlings were decanted and concentrated in an atmosphere of nitrogen to prevent 52 oxidation of possible indole compounds. These concentrates were then made up to a volume of 50 milliliters with ethanol. The subsequent concentration, procedures Extract 1: separation and chromatographic utilized were as follows: Ten milliliter aliquots of the 50 milliliter ethanol extracts were concentrated on a steam bath to one milliliter and chromatographed as Extract 1 (Table XI). Extract 2: Although seedlings were grown in the dark and little or no chlorophyll was extracted a water soluble pig­ ment, probably a carotene, was obtained which often caused streaking on the filter paper. the desired compounds, To separate pigments from portions of Extract 1 were spotted heavily on filter paper sheets which were then developed with water. The pigmented spots were then cut off and the remainder of the sheets extracted with ether for 10 hours. These ether extracts were concentrated to 1 milliliter and chromatographed ( 4 4 ) , Extraction of Frozen Se edli ngs : The seedlings placed at -5°C were next thawed and extracted with peroxide-free ether and sand in the manner previously described for the extraction of fresh seedlings. These ether extracts were treated as fol­ lows to provide Extracts 3 to 8 (Table XI): Extract j}: Portions of the original ether extracts from fro­ zen seedlings were chromatographed. 53 Extract 4: The remainder of the original ether extracts were concentrated to 50 milliliters and chromatographed. Extract 5: Ten milliliters of Extract 4 were concentrated to 1 milliliter and chromatographed. Extract 6: was Another 10 milliliter aliquot from Extract 4 spotted heavily on filter paper, developed with water, dried and the pigmented spots cut off. The remaining por­ tion of the paper was then extracted 10 hours with ether. The resulting extracts were concentrated and chromatographed. Extract Pigments in Extract 5 were separated in the manner described for Extract 6. The resulting ether extracts were concentrated and chromatographed. Extract 8: Twenty milliliters of Extract 3 were shaken with sodium sulfate to remove water and to salt-out adsorbed compounds. The clear solutions were concentrated and chroma­ tographed. Chromatographic Techniques: Standard ascending and descending chromatographic techniques were used for the separation of indole compounds. Seed extracts, together with Indoleacetic Acid and Tryptamine at known concentrations, were spotted on filter paper sheets, air dried and developed. The developing agents used were those described by Nitsch and included water, (44) (a) Isopropanol - 28 per cent ammonia water - (8:1:1 V/V), and (b) water. Salkowski's (FeClg) and 54 E h r l i c h ’s (p-Dimethylaminobenzaldehyde or PBZ) reagents, and a 2537 A ultra-violet light source were used as detecting agents after the filter paper was air dried. colored Relatively few spots were obtained from ether extracts of vernalized lettuce seedlings perhaps indicating (1) an extremely low concentration of indole compounds in the seedlings, too low a concentration of indole compounds or (2) in the extracts for detection by the methods employed. Rf values of the IAA and Tryptamine controls approxi­ mated 0,36 and 0.73 when developed with Isopropanol-ammonia water-water and 0.23 and 0.86, with water. respectively when developed Possible naturally occurring indole compounds are indicated in Table XI. Colored spots were found at Rf 0.63 on chromatograms of Extract 4 of vernalized seedlings developed with isopropanol-ammonia water-water with Salkowski's reagent), (detected and at 0.83 on chromatograms of Extracts 6 and 7 (vernalized seedlings) developed with water and detected with PBZ. with ultra-violet light. All other Rf values were obtained In Extract 1, while no Rf values were obtained by chromatographing ether extracts of vernalized seedlings, some comparable to those of 3 - Indoleacetic Acid, and Tryptophan occurred consistently (ultra-violet light) the non-vernalized seedling extract developed both with propanol-ammonia water-water and with water in iso­ (Table XI). values approximating those for Indoleacetonitrile and the Rf 55 Ethyl Ester of Indoleacetic Acid were also found on chro­ matograms of some extracts. Significantly, several of the extracts of non-vernalized seedlings developed with Isopropanol-ammonia water-water provided R^ values around 0.52, at which no naturally occurring indole compounds have been detected insofar as the author is aware. Only in Extract 6 was an Rf of 0.52 obtained on a chromatogram from a verna­ lized seedling extract. Second Seed L o t : A further study of indole compounds was initiated utilizing a new lot of vernalized and non-vernalized seed. V e r n a 1izina S e e d : chromatograms The paucity of colored spots on in the previous study suggested that a higher concentration of indole compounds was necessary. vernalized. Therefore, in the original extract additional trays of seedlings were To cireurnvent the lack of uniform germination obtained previously, germinated per tray. only 75 grams of lettuce seed were A layer of wax paper was placed between the tray and the filter paper to facilitate removal of the seedlings. Vernalization was started June 23 and control seedlings were germinated July 12. zen at -5°C on July 15. All seedlings were fro­ About three kilograms of seedlings (fresh weight) were obtained after thawing. Extraction of Frozen Seedlings: Methods similar to 56 TABLE XI Rf VALUES OF POSSIBLE NATURALLY OCCURRING INDOLE COMPOUNDS SEPARATED BY PAPER CHROMATOGRAPHY FROM ETHER EXTRACTS OF VERNALIZED AND NON-VERNALIZED GREAT LAKES LETTUCE SEEDLINGS (DETECTION BY ULTRA-VIOLET LIGHT) Extract No 1 2 Dftvfi]ope r .. Likely Indole / S eed Isopropanol- water Treatment ammonia C ompound water-water (8:1:1 V/V) (Rf Valu ps) Separation Procedure (First Seed Lot) Ether extraction of Fresh Seedlings:! ml. ethanol extr. from original ether extr. Ether extr. of Extr. 1 pigments removed. V* None None NV * 0. 24 0.34 0. 56 0.85 V None None —— 0. 50 0.55 None ? V None 0.52 NV 0.49 to 0. 56 None ? do IAA NV 3 4 Ether extr. of Fro­ zen seedlings: Or ig i na1 extr. Extr. 3 conc. 50mls . to V NV ^ 0.34 0. 63** None — T r y p .* * IAA Tryp. do IAA Tryp. 5 V 10 ml. aliquots of NV Extr. 4 conc. to 1 ml. 0.34 0. 28 0.90 0. 58 6 10 ml. aliquots of Extr. 4, pigments removed V NV 0. 52 0.90 None ? 0.6344 IAA None Extr. 5, pigments removed V NV do do 7 0. 83 444 IAA 57 TABLE XI Extract No. Procedure (First Seed Lot) (Continued) Peveloper Seed Isopr opanol- water Treatment ammonia water-water Likely Indole C ompound (RfValues) 20 ml. aliquots of Extr. 4 dried with N a 2S 04 and conc. V NV 0.77 0.80 0. 37 0. 52 None 0.91 IAN ET IAA IAA *V and NV indicate Vernalized and Non-verna1ized **Tryp., IAA, IAN and ETIAA are tryptophan, 3 - Indoleacetic Acid, 3-Indoleacetonitrile, and Ethyl Ester of Indoleacetic Acid, respec t ively. 4 Brown - Detected with Salkowski*s Reagent “^ P u r p l e - Detected with Ehrlich's Reagent ^ ^ G r a y - Detected with Ehrlich's Reagent 58 those previously described were utilized in the extraction of indole compounds from the second lot of seedlings. The following concentrates and separations were made: Extract 1: Portions of the original ether extracts were chromatographed Extract 2: (Table XII). The remainder of the original extracts were next concentrated and made up to volumes of 100 milliliters with water and chromatographed. Extract The water layer from Extract 2 was separated* acidified to pH 2.85 (59), re-extracted with ether, ether extract concentrated to 1 milliliter. and the This was chro­ matographed. Extract 4: Extract 3 was next spotted on filter paper sheets which were then developed with water. The pigmented spots were cut off and the remaining portions of the sheets extracted with ether. These ether extracts were concentrated and chromatographed. Extract J5: Another method for the removal gested by Nitsch (44) was employed. of pigments sug­ Ten milliliter aliquots of Extract 2 were extracted 24 hours by 1iquid:1iquid par­ tition with hexane to remove lipids and pigments and with acetonitrile to dissolve auxins. The hexane fraction was then concentrated and chromatographed. Extract 6,: The acetonitrile fraction was heated to dryness on a steam bath and the residue dissolved in a small quantity 59 TABLE XII R f VALUES OF POSSIBLE NATURALLY OCCURRING INDOLE COMPOUNDS SEPARATED BY PAPER CHROMATOGRAPHY FROM ETHER EXTRACTS OF VERNALIZED AND NON-VERNALIZED GREAT LAKES LETTUCE SEEDLINGS (DETECTION BY ULTRA-VIOLET LIGHT) Extract No. 1 2 3 4 Separation Procedure (Second Seed Lot) Seed Treatment 6 Likely C ompound Original ether extracts of frozen seedlings v** NV** None do — Extr. 1 conc. and made to a volume of 100 mis. with water V 0. 23 0. 42 0. 24 0. 57 0.84 ? IAN* ? Tryp. IAA Water layer from Extr. 2 separated, acidified to pH 2.85, extr. with ether. Ether extr. conc to 1 ml. Extr. 3, pigments removed NV V • NV Extr. 2, pigments re­ moved by liquid:liquid partition Hexane frac­ tion Extr. 2, pigments re­ moved by 1iquid:1iquid partition Acetonitrile fraction: 0.2644 0.39 ° ’4? 0.58 0. 25 0.39 0* SI 0.63 ? IAN ET IAA to Tryp. ? IAN f o, Tryp. o ET IAA 0.7844 0.69 0.7844 ? IAA ? V 0. 55 Trp. NV None V 0.37 0.52 0. 23 0.42 0. 57 V NV 5 Rf *** Values NV IAN Tryp. ? IAN ET IAA *IANf Tryp. IAA, ET IAA are 3- Indoleacetonitr il'e, Tryptophan, 3 - Indoleacetic Acid, and Ethyl Ester of Indoleacetic Acid, respectively. **V and NV indicate Vernalized and Non-Vernalized ***Developed with water •♦•♦Blue - detected with Ehrlich's Reagent 60 of ether. These ether solutions were then chromatographed. Chromatographic techniques were identical with those already described. Values from extracts of lettuce seedlings comparable to compounds occurring naturally are indicated in Table XII. With water as developer colored spots appeared at Rf values of 0.26 in Extract 3 (vernalized seedlings), and at 0.77 and 0.78 of Extract 4, both from vernalized and non-vernalized seedling extracts. Rf values possibly indi­ cative of Indoleacetonitrile, Tryptophan, Indoleacetic Acid, and Ethyl Ester of Indoleacetic Acid were obtained. no specific differences However, in biochemical substances resulting from vernalization were discerned. Determination of L-Trvotophan: Vernalizing S e e d : Four one-hundred gram lots of lettuce seed were spread evenly on layers of moist filter paper in two large stainless steel trays. These were held at 60°F for 48 hours and then shifted to a 40°F temperature for 18 days. On July 4 two additional trays of seed were prepared and held at 60°F until July 7 when all seedlings were frozen at -5°C. Extraction and Determination of Fjee L-Trvptophan: According to the method described by Nitsch tryptophan (44) free in lettuce seedlings was determined quantitatively by precipitating proteins with boiling ethanol for three minutes, evaporating the alcohol on a steam bath, extracting 61 tryptophan twice with hot water, and shaking the aqueous extract with ether to purify it of indole and anthranilic acid which give positive tests with the bioassay. of the aqueous extract was adjusted to 6.0. The pH Tryptophan was measured by employing Lactobaci11 us arabinosis1 as a bio­ ass . Quantitative determination of the free tryptophan in the water extracts revealed that vernalized seedlings yielded .00127 per cent and non-vernalized seedlings .00293 per cent L-tryptophan on a fresh weight basis. The author wishes to thank Dr. Erwin J. Benne and Dr. Richard W. Luecke, Department of Agricultural Chemis­ try, Michigan State University, for determining total ni­ trogen (necessary to establish standard curves for the bioassyX and for performing the bioassay, respectively. 62 DISCUSSION ENVIRONMENTAL FACTORS AFFECTING FLOWERING OF LETTUCE Indices of F 1owerina: The number of leaves or nodes preceding the developing inflorescence is recognized as a specific 81). index of flowering (23, 34, 36, 37, 40, 47, 80, It is especially useful in vernalization studies with lettuce because it is associated with floral initiation in response to various environmental conditions. a "physiological strated index" like leaf numbers is best demon­ in the growth regulator studies (Table V) and in the 1954 and 1955 night temperature studies VII). The value of (Tables VI and The application of Maleic Hydrazide to seedlings dur­ ing vernalization inhibited vegetative growth during early plant development at 70°F and resulted in a significant delay in days to flowering. However, the fact that leaf numbers were reduced preceding a distinct developmental stage (e.g. the developing inflorescence) indicates a speci­ fic acceleration of flowering by the chemicals. In Tables VI and VII, while 27.6 and 34.8 leaves preceded the appearo ance of the developing inflorescence at 70 and 65 F, respectively, 109 and 107 days preceded the same stage. The acceleration of flowering at the higher temperature, indicated by leaf numbers, as is not supported by the number of 63 days to the appearance of the visible flower parts. Ver­ nalized plants grown at 70°F under similar conditions in 1954 and 1955 produced 27.6 and 27.7 leaves during the two years, respectively. In contrast, plants at 65°F in 1955 produced 3.5 fewer leaves than those grown in 1954. It is suggested that the difference probably resulted from addi­ tional light received by those plants grown in the 1955 experiments. Further evidence for the reduction in leaf numbers preceding the developing inflorescence plants) (vernalized by extended photoperiod occurs in Table X. With a 16-hour day a difference of only two leaves preceded the developing inflorescence of plants grown at 60 and 70°F night temperatures. Leaf numbers of plants grown at the same temperature varied between experiments. ences This was probably due to differ­ in season of planting (naturally occurring photoperiods), the effects of additional lighting, and even growing plants in different pot sizes or in a ground-bed. Despite the vari­ ation between experiments similar imposed factors, temperature, such as increased or decreased leaf numbers relatively within each experiment. In the 1954 night temperature experiment (Table VI) seedstalk elongation was utilized as an index of flowering (1). Although the initial effects of temperature on seedstalk development were clearly indicative, as growth progressed the 64 differences between vernalized and non-vernalized plants decreased until, (1) finally, they no longer existed. in comparable observations, Andrew found that the effects of chemical growth regulators on elongation and branching of the inflorescence were not indicated by measurements of seed­ stalk heights. development, Except where growth regulators delayed plant coupled with such complementary indices as the production of seedstalks without previous head formation and the number of days preceding visible flower parts and flower­ ing, leaf numbers serve as a reliable index of reproductive express ion. The results suggest that the major factors affecting flowering of vernalized lettuce are temperature and photo­ period. A discussion of these factors, therefore, may ex­ plain many of the negative results obtained in the present as well as in other studies. Night Temperature: (15, 22, 23, Flowering in a variety of crops 24, 33, 35, 36, 39, 43, 47, 51, 57, 68, 72, 73) has been hastened by cold-exposure of seedlings or young plants. The results of the night temperature experiment indicate a critical thermoperiodic specificity for flowering in lettuce grown from vernalized seedlings. tion of flowering in lettuce with high, The accelera­ and the delay with low night temperatures following vernalization is both of fundamental and practical interest. Under greenhouse tempera­ 65 tures favorable for normal vegetative development 6 0 ° F ), regardless firm heads. of vernalization, (50 to lettuce plants formed In contrast, with high night temperatures seed­ stalk development was promoted in vernalized lettuce plants. In the 1954 night temperature experiment no differences in flowering were found in vernalized plants grown at 65 and ° o 70 F after transfer from 50 and 65 F, respectively, suggest­ ing that temperature conditions during early plant growth do not affect subsequent flower expression of vernalized lettuce pla nts. To determine the specific effects of cool and warm temperatures in delaying and accelerating flowering, vernali­ zed and non-vernalized plants were grown at 50 and 70°F for o 66 days and then interchanged. Exposure to 70 F night temper o ature induced seedstalks following 66 days at 50 F while flowering was delayed when warm preceded cool temperatures (Table VIIIX In comparison to non-vernalized plants shifted to 50°F from 70°F, directly. vernalized plants produced seedstalks Seemingly, in lettuce the potential for flowering is greater following vernalization, but expression is delayed by subsequent 50°F night temperatures. Photoperiod: Photoperiod, with night temperature, nalized head lettuce. separately and interacting markedly affected flowering in ver­ A 16-hour day promoted and a 9-hour day delayed seedstalk development without prior heading 66 irrespective of 60 or 70°F night temperature. Although plants grown with a short day flowered after initially forming firm heads, of vernalized, the number of days preceding anthesis as compared to non-vernalized plants, were not significantly different. There are several reports 70), many of them contradictory, (1, 2, 4, 7, 8, 53, 68, on the effects of photo­ period and temperature on flowering of lettuce. In a brief note on photoperiod and temperature studies with vernalized lettuce, Milthorpe and Horowitz (42) reported that high temperatures and long days promoted flower­ ing. However, this report included no experimental data nor did it describe the treatments used. Andrew (1), using 9 and 15-hour photoperiods and 50 and 60°F night temperatures found that long days and high temperatures affected early seedstalk development. however, He obtained accelerated flowering, o under conditions of 50 F and short days and con­ cluded that "neither factor - (temperature or photoperiod)appeared to be the limiting or controlling influence in determining vegetative or reproductive development". was It observed in the present study that with 50°F night temper­ atures and continuous 24-hour photoperiod, nalized, seedstalks in ver­ as compared to non-vernalized plants, were induced. Analysis of A n d r e w ’s methods indicated that the experimental plants were "pricked out" March 1, and potted April 8. 67 Measurements July 14, of seedstalks were recorded from June 4 to Thermograph records in 1954 and 1955 indicated that although 60°F night temperatures could be maintained until mid-June, after March 1 maintenance of 50°F night temperatures within the greenhouse was not possible. As indicated by leaf numbers preceding the develop­ ing inf1 orescence, with and days to anthesis (Table IX), only long days were 60°F temperatures effective in promoting seedstalk development. With short days lettuce plants at 60 and 70°F formed firm vegetative heads irrespective of vernalization. To determine the specific effects of short days on flowering of lettuce, heads of vernalized plants grown at 70°F and a nine hour day were cut open and fortyfive leaves were found to precede the developing inflores­ cence of the enclosed seedstalks. This indicates that short days actually delay expression of flowering of vernalized plants grown at high temperatures. That vernalized and non­ vernalized lettuce plants flowered simultaneously when grown with a short day and a cool temperature suggests that the effects of vernalization were nullified (Figure 6). The critical temperature and photoperiod requirements subsequent to chilling of lettuce seed for acceleration of flowering in the greenhouse, were verified in field studies. Overwintered seed and seedlings and vernalized and non-ver- nalized transplants placed in the field in the early Spring 68 all formed marketable heads. Thermograph records revealed that average night temperatures in the vicinity of the ex­ perimental plots remained below 60°F. The remarkable sensitivity to cool temperatures of Full Heart Batavian endive seed is of economic signifi­ cance since vernalized endive plants flowered two months earlier than those not vernalized. market grower therefore, It would behoove a to delay planting endive at least until May 1 to prevent cold-induetion. On the other hand, by early transplanting or direct seeding, planting of vernalized duced in the West, <; n i plants, or late trans­ endive seed, normally pro­ could perhaps be produced in Michigan. l Te mperature: vernalization stimulus It has been reported that the is perceived by the embryo and likely the apical meristem (48). (25, 26) A change from the vege tative to the reproductive condition in the apical meristem, after chilling JMatthiola plants four days, was reported by Emsweller and Borthwick therefore, (15). It is especially interesting, that subsequent to vernalization different air temperatures significantly altered the number of leaves pre­ ceding the developing inflorescence, were not affected by root but that leaf numbers (soil) temperature. This may support the view that the apical meristem is the site of perception of the vernalization stimulus. Since leaf numbers were not influenced by root 69 temperature (e.g. "physiological" as contrasted to "chrono­ logical" reproduction was not accelerated) high soil tempera­ tures seemingly affect the rate of seedstalk development rather than the response to vernalization per se . THE VERNALIZATION PROCESS The previous discussion of environmental factors affecting flowering of lettuce contains the information necessary for evaluation of the experiments concerned with the vernalization process. Duration of Chil lin g: The duration of cold exposure necessary for acceleration of flowering in lettuce has been previously investigated (1, 24, 33, 61). Knott (33) found that 10 to 20 days at 40°F was sufficient to accelerate seed­ stalk development; Simpson the longer period was more effective. (61) and Andrew (1) concluded that 16 days chilling effectively vernalized the varieties Great Lakes, and Slobolt. 56 days was shown by Gray Ideal, Imperial 847, Exposure at 40°F for 28, 42 and (24) to promote flowering in lettuce equally although the longer periods of chilling weakened the seedlings. The present results indicated that a minimum of 13 days of vernalization is necessary for Great Lakes lettuce. Age of the plant: Another factor influencing the response of lettuce to cold exposure is the age of the plant 70 or the stage of development. Reimers lettuce exposed to temperatures leaf, With Warne (79) found that of 2 to 5°C at the first in contrast to germinating seeds, flowered later. seed germinated 24 and 72 hours prior to vernalization (76) found that seed with radicles emerged was not receptive to vernalization. were generally (72 hours) Andrew's resu,lts (1) in accord with those of Reimers. He concluded that there was no appreciable difference between cold expo­ sure of seed at the swollen stage and after the radicle emerged. Andrew further reported that seedlings two to three centimeters smaller seedlings, seedlings, (76), in height were set back, in comparison to by subsequent cold temperatures. contrary to the results of Reimers Such (79) and Warne produced plants which flowered earlier than non-ver- nalized plants. In the present experiment, plants germinated 72 hours or more prior to cold exposure were delayed in flowering as indicated by leaf numbers prior to the appearance of the developing inflorescence. It is interesting that the number of days to anthesis tended to decrease in relation to a progressive Lacking increase in days of germination prior to chilling. in the experimental design were treatments with plants germinated 0, 24, and 48 hours. observations However, subsequent in the present study indicated that even seed germinated 40 hours were effectively vernalized. 71 The acceleration of flowering by vernalization without pre­ soaking seed, described by Andrew (1), was also observed in this study. Temperature Patterns During V erna1ization: cussing the results of Thompson and Kosar Dis­ (69), Andrew (1) suggested that the lack of response of Slobolt and Cos lettuce may have resulted from temperature (a) exposure of the seed to too cold a (1 to 2°C ), (b) exposure of the control seed to low temperatures for a "few hours" which may have had a slight vernalizing effect. The fact that chilling seed at 32°F did not stimulate flowering lends credence to Andrew's suggestion (a), above. The apparent lack of information concerning the temperature factors during vernalization which affect flower­ ing suggested a series of detailed temperature studies. indicated in the results, As irrespective of 35, 40, or 45°F temperature patterns during vernalization, firm heads when grown at 50°F temperatures. the plants formed Since at this temperature no seedstalks developed the delaying effects on flowering of 50°F temperatures are further emphasized. The effects of high temperature interruptions of the chilling period are shown in Table III. plants vernalized vernalization, In comparison with those in the usual manner or interrupted during seed germinated at 60 or 90°F and grown subse­ quently at 65°F night temperature produced plants which were delayed in flowering. Lack of uniformity resulting from root 72 rots may have been responsible for the relatively small difference in days vernalized plants. to anthesis between vernalized and non­ The results of this study suggest that a critical time period may exist in lettuce seed vernalization during which cold-exposure is effective in accelerating flower­ ing. The detection of such a period would materially aid in­ vestigators in the biochemical isolation of substances associated with the vernalization stimulus. Moisture During Vernalization: of rye (47) and mustard (57), In the vernalization prior to cold-exposure, seed was pre-soaked sufficiently to cause swelling but not emergence of the radicle. Mustard seed vernalized in this manner was then dried and stored as long as six years without loss of the accelerating stimulus. produces radicles Since lettuce seed germinated at 40°F it was necessary to modify the pre-soaking and drying techniques utilized. The results indicate that seedstalk development was accelerated only from seedlings pre­ germinated 24 hours and vernalized in the usual manner. Soako ing 6 and 12 hours and drying before exposure to 40 F, or soaking 24 hours at 70°F prior to chilling at 32°F did not accelerate flowering. Additional studies aimed toward obtain­ ing non-sprouted vernalized seed are indicated. Perhaps pre­ soaking for 12 hours and maintaining seed at 40°F in a high atmospheric humidity may permit satisfactory vernalization without the emergence of radicles. 73 Light Quality and Dura tion: The effects of light quality on lettuce seed germination have been studied for many years with (5, 6, 17, 38). In the present study the delay light from 100 watt mazda lamps at 40°F offers an in­ teresting cool temperature/light interaction worthy of further study. In 1951 Vogt Indoleacetic Acid violet light. (75) reported the destruction of 3- in the roots of cereal crops with ultra­ Terpstra (65) trtrt-ained comparable results with oat coleoptile tips. The effects of ultra-violet light on seedlings before and during chilling were studied* therefore to associate indirectly the effects of auxins and vernaliza­ tion on subsequent floral initiation. The plants from ir­ radiated seedlings were transferred to the field August 16. Possibly because of cool night temperatures and decreasing daylengths during September and October, seedstalk develop­ ment was prevented and only firm heads were formed. The recognized influence of photoperiod on plants suggested that duration of light during germination might also affect flowering. When plants were subjected to dif­ ferent daylengths during vernalization, only heads were formed. however, at 50°F This is further evidence for the predominant effects of night temperature on flowering irre­ spective of treatment during vernalization. Chemical Growth Regulators: Growth regulator appli- 74 cations during and subsequent to seed germination and ver­ nalization have influenced flowering 14, 79). in lettuce (1, 12, 13, A series of experiments were performed to evaluate the effects of Maleic Hydrazide and 2,4-Dichlorophenoxyacetic Acid and of various other growth regulators on flowering in lettuce. Again, plants grown either at 50°F or in field plots when night temperatures were conducive to head formation, mained vegetative. temperature re­ The primary effectiveness of high night in accelerating flowering of vernalized lettuce was displayed in a study of the effect of 60 and 70°F night temperature on plants from seedlings vernalized together with o 20 or 40 ppm of MH. Only those plants grown at 70 F produced seedstalks without head formation irrespective of growth regu­ lator treatment. effective Only at the higher temperature was MH in reducing leaf numbers preceding the developing inf1 orescence. Andrew (1) concluded that differences response to the same growth regulators could to one or more factors: (a) concentration, logical age" of the treated plants, from seeding. However, in flowering be attributed (b) the "morpho- (c), the number of days "the factor or factors affecting the degree and type of influence of growth regulators on the seedstalk development of lettuce is as yet uncertain". The results of the present investigation suggest that environment, subsequent to seed treatment or growth regulator application 75 to plants, is a primary factor which must also be considered. In relation to the results of Franklin (1), and Clark and Wittwer (13) Lakes and Cornell 456 plants (18), Andrew it is interesting that Great in a field experiment with growth regulator application during and/or subsequent to vernalization formed firm vegetative heads when transplanted May 21, 1954. Apparently these factors were not effective in overcoming the influence of cool night temperatures pre­ venting flowering of lettuce. Franklin (18) In a recent communication indicated that the stage of development for spraying was so critical that only during a delimited period of growth, measured in heat units, were 2,4-D sprays effec­ tive in accelerating seedstalk development in lettuce at Parma, Idaho. It appears that the factors which normally stimulate flowering in lettuce are even more effective when operative in combination with vernalization. of high night temperatures, Seemingly, a combination high root temperatures, and long days contribute to acceleration of flowering and seedstalk development in head lettuce grown from vernalized seedlings. 76 BIOCHEMICAL STUDIES Using paper partition chromatography for the separa­ tion of naturally occurring indole compounds in ether extracts of vernalized and non-vernalized lettuce seedlings, which fluoresced in ultra-violet were detected. many spots Several ap­ proximated those for compounds known to occur in plants. However, no single Rf value was found peculiar to chromato­ grams of either vernalized or non-vernalized lettuce seed extracts (Tables XI and XII). This may indicate that none or all of the compounds obtained are associated with the stimulative process. As suggested by the appearance of only a few colored spots with Salkowski's and Ehrlich's reagents on chromatograms of the extracts from lettuce seedlings, the concentration of indole compounds may have fallen below the range which is detectable by the chromatographic technique, thus preventing characterization of such compounds occurring in extremely minute quantities. Although many values com­ parable to those of indole compounds occurring in plants were obtained by chromatographing seedling extracts on filter paper, it is possible that many resulted from lipids or were artifacts. This suggests the need for a bioassay and/or quantitative chemical analysis in conjunction with chromato­ graphic separations to ascertain and relate specific biological activity to biochemical differences induced by vernalization 77 of lettuce seed. L-tryptophan The significantly low percentage of in vernalized, as compared to non-vernalized seed suggests direct utilization of L-tryptophan or con­ version to another substance, Acid during vernalization, differences conceivably 3-Indoleacetic or may be indicative of other likely to occur in the auxin complex in the seedlings as a result of vernalization. 76 SUMMARY AND CONCLUSIONS Seed vernalization, daylength, plant growing temperatures, and chemical growth substances, separately and interacting, markedly influenced flowering and seedstalk development in head lettuce as indicated by leaf numbers and days preceding the inflorescence and days to anthesis. A minimum of 13 days at 40°F was required for ver­ nalizing moist lettuce seed pre-germinated less than three days at 60 to 70°F. the usual manner, drying, In comparison to seed vernalized in soaking with water for 6 or 12 hours, then holding at 32 or 40°F was not effective. temperature (60 or 90°F) High interruptions of vernalization for two hours daily or at intervals of three days did not pre-* vent subsequent acceleration of flowering. Seed vernalization together with 20 or 40 ppm of Maleic Ilydrazide resulted in plants which flowered after the appearance of fewer leaves (nodes) than those from seed vernalized with water alone. Night temperatures above 65°F subsequent to seed vernalization accelerated flowering and resulted in seed­ stalks without preceding head formation. Below 65°F vernalized plants first produced a high percentage of firm, vegetative heads and then flowered. Non-vernalized plants flowered only at night temperatures above 65°F. Earlier flow­ 79 ering resulted in plants exposed continuously to warm tempera­ tures as compared to alternating cool night temperatures. Apparently, (50°F) and warm (70°F) the potential for flowering is greater following vernalization but a critical temperature for plant growth exists for reproductive expression. Photoperiod, temperature, lettuce. separately and interacting with night markedly affected flowering in vernalized head The minimum night temperature following vernaliza­ tion at which early flowering was favored was reduced from 65 to 60°F when plants were grown at a daylength of 16 hours, o At 60 F with a 9-hour photoperiod, both vernalized and non­ vernalized plants produced firm heads which flowered simul­ taneously, factors suggesting that this combination of environmental prevents expression of the seed vernalization s t imulus. Root (soil) temperatures seedstalk development influenced the rate of (measured as days to anthesis) rather than the expression of vernalization (leaf numbers preced­ ing the appearance of the developing inflorescence). This supports the supposition that the apical meristem perceives the vernalization stimulus. (64 and 70°F) temperatures flowering At high soil temperatures in days was promoted, while at low (50 and 57°F) flowering was delayed. Field studies with vernalized lettuce verified the controlling effects on flowering of photoperiod and tempera­ 80 ture critically demonstrated with house. days, plants grown in the green­ For seed production the practical utilization of long high night temperature, and high root temperature which accelerate seedstalk development in vernalized lettuce lies in the selection of appropriate planting dates or manipula­ tion of the factors in the greenhouse for increasing breeding stock during the Winter. Conversely, head lettuce for market should be produced when night temperatures average below 60°F. By early Spring transplanting or direct seeding, late transplanting plants, or of vernalized Full Heart Batavian endive flowering was accelerated by two months. This sug­ gests that endive for market should be planted after May 1, but for seed production April in Michigan, direct seeding in early is desirable. In biochemical and non-vernalized violet light) studies, with extracts of both vernalized seed, a series of fluorescent spots (ultra­ and occasionally colored spots (normal light were separated by paper partition chromatography. Such extracts re­ peatedly yielded fluorescent spots comparable to those of 3Indoleacetic Acid, Tryptophan, Ester of Indoleacetic Acid water Indoleacetonitrile, and Ethyl in both water and isopropanol-ammonia (6:1:1 V/V) and water systems. Fluorescent spots also occurred on chromatograms of both vernalized and non-vernalized seed extracts at an Rf value of 0.55, where no biologically 81 active substances have been reported insofar as the author is aware. vernalized, A lower percentage of L-tryptophan was found in as compared to non-vernalized seedlings, ing that during seed vernalization L-tryptophan is either utilized directly or converted to another compound, able 3 - Indoleacetic Acid. suggest­ conceiv­ 02 LITERATURE CITED 1* Andrew, W. T. 1953. Seedstalk Development of Lettuce as Affected by Growth Regulators, Vernalization, Temperature and Photoperiod. Doctoral Thesis. Michigan State College. 2. Arthur, J. M. and J. D. Guthrie, 1927. Effect of Light, Carbon Dioxide, and Temperature on Flower and Fruit Production. Mem. Hort. Soc. N. Y. 3:73-74. 3. Avery, G. S., J, Berger, and B. Shalucha, 1942. Auxin Content of Maize Kernels During Ontogeny from Plants of Varying Heterotic Vigor. Amer. Jour* Bot. 29: 765-772. 4. 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