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EFFECTS OF VISIBLE MONOCHROMATIC RaDIHTIONS ON GROWTH
OF PITH CALLUS TISSUE OF FBLAKGQNIUM ZONALE
APPROVED:
Major Professor
(J.UU Minor Professor
^ VAy -\a 1 V-1 A
Director of the Department of Biology
Dean of the Graduate School
EFFECTS OF VISIBLE MONOCHROMHTIC RADIATIONS ON GROWTH
OP PITH CALLUS TISSUE OF PELARGONIUM ZONAL£
THESIS
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirement s
For the Degree of
MASTER OF SCIENCE
By
H. Bailey Ward, B. S
Denton, Texas
August, 1966
TABLE OF CONTENTS
P age
LIST OF TABLES iv
LIST OF ILLUSTRATIONS v
Chapter
I. INTRODUCTION . I
II. MATERIhLS AND METHODS. 10
III. RESULTS 18
IV. DISCUSSION 26
BIBLIOGRAPHY 33
ill
LIST OF TABLES
Table Page
I. Composition of White's Modified Medium 11
II. Initial and Final Weights of Callus Tissue of Pelargonium zonale Variety Enchantress Flat Grown in Five Different Light Treatments 19
III. The Significance of Different Light Treatments on Mean Fresh Weight Increases of Cultures of felarffpnluBSj zonule. Grown Ij, Vitro as Determined by the Randomized Block Design for Analysis of Variance 24
IV. Significant Differences of Mean Fresh Weight Increases of Callus Tissue of Pelargonium zonale Between Individual Light Treat-ments as Determined by the Student!zed Range Test. . 25
iv
LIST OF ILLUSTRATIONS
figure Page
1. Diagram of the Tissue Culture Chamber 16
2. Fresh Weight Increases (Per Cent Over Initial) of P.el.ar.qo.aiam Callus Tissue Grown Under Different Light Treatment 20
3. Photomicrographs of Callus Tissue of Pelargonium 22
CHAPTER I
INTRODUCTION
In the early 1920's, Bobbins (27) and Bobbins and Maneval
(28) succeeded in growing excised plant roots for several weeks
in a defined nutrient solution. However, it was not until
1934 that the field of plant tissue culture was opened up by
two workers, White and Gautheret (6) . White (43) had empha-
sized the usefulness of growing excised tomato roots jji vitro
for extended periods of time. These were the first success-
ful cultures of plant organs. A few years later, White (44)
published descriptions of the first successful cultivation of
plant tissues. These early cultures represented the first
true plant tissue cultures in the sense of cultures of un-
organized plant rnateri als.
Before long-continued cultivation of plant tissues grown
in vitro was demonstrated, the development of tissue culture
technique as a useful method in botanical research was out-
lined by White (41). Additional reviews concerning materials,
techniques, and history have been presented by White (39, 40,
41, 42, 45), Gautheret (7, 9), Happaport (25), Street (36),
Hiker and Hildebrandt (26), Butcher and Street (2), Heller
(10), Narayanaswarai and Norstog (21), Tulecke (37), and
Hildebrandt, Hiker, and Duggar (11).
A number of different media h a v e been developed for the
nutrition of plant tissue cultures. Many of these are entirely
synthetic (9, 10, 23, 45) while others contain basic mineral
elements corabined with such ill-defined substances as yeast
extract, coconut milk, or tomato juice. In attempts to ana-
lyze coconut milk, Pollard, Shantz and Steward (24) and Shantz
and Steward (29) revealed growth-promoting s u b s t a n c e s w h i c h
stimulated the growth of carrot phloem explants. Steward and
Caplin (35) found s y n e r g i s t i c effects between 2,4-dichloro-
phenoxyacetic acid and coconut milk after f i n d i n g that a cora-
bi nation of the two was necessary for growth of potato-tuber
tissue grown in vi tro. Sirailar s y n e r g i s m s have been reported
between i ndoleaceti c acid and ki neti n (6-furfuryl ami nopuri ne)
(32), and 2,4-dichlorophenoxyacetic acid and ki netin (1).
Skoog and Miller (31) demonstrated the role of ki neti n in the
regulation of root, callus, and shoot growth in tobacco pith
tissue.
The n u t r i t i o n a l requirements of plant tissue cultures
have thus been generally e s t a b l i s h e d . The search for better
media, new g r o w t h - p r o m o t i n g substances, and d e f i n e d culture
c o n d i t i o n s is at present in an active state.
The usefulness of plant t i s s u e c u l t u r e s in studies of
disease, nutrition, and m o r p h o g e n e s i s has been lim i t e d by the
relative i n s t a b i l i t y of the growth of plant t i s s u e s in jjj,
vitro cultures. N u t r i t i o n a l requirements (10), morphological
and cytological form (3), and g e n e r a l organizational a b i l i t i e s
of plant tissue cultures often change with the passage of
time. Although in any studies have been made in an at temp t to
determine optimal nutritional requirements (10, 12, 33, 40)
and temperature regimes ( 4, 5, 11), little is known about the
influences of visible radiation on the growth and development
of plant callus tissue. Much of the research being done with
light-induced m o r p h o g e n e s i s , or photomorphogenesi s, has in-
volved the use of intact plants or plant organs. It is at
this level that plant tissue cultures may be effectively used.
De Capite (4) studied the effects of different intensities
of artifici al light on callus t i s s u e growth but f a i l e d to
specify whether the source of radiation was incandescent or
f l u o r e s c e n t . W h i t e ( 4 5 ) and White and Risser (46) simply
stated that cultures should be i n c u b a t e d i n the light or i n
darkness. Early in the history of plant tissue culture,
La Hue (15) noted differences in root formation abilities of
hypocotyls and cotyledons from various species when grown In
the light and in darkness. Some workers have reported that
respiration rates and differentiation were promoted by light
( 5 , 8 , 2 0 ) . Nickel1 al. (22) demonstrated that t h e g r o w t h
of some tissues may be stimulated by light. However, Stein-
h a r t al. (34) reported that the growth of s p r u c e tissue
cultures was more pronounced in d a r k n e s s than i n l i g h t .
It has been amply demonstrated that specific wavelengths
of v i s i b l e radiations are factors i n plant m o r p h o g e n e s i s .
The red portion of the visible spectrum has been implicated
4
in several morphological responses. The phytochrorae system,
reversibly responding to red (660 mu) and far-red (730 mu) ,
has perhaps been given more attention than any other system.
Liverman and Bonner (16) reported on the reversibility of the
phytochrorae system and Lockhart (17) has studied the system
in relation to physical growth factors. Lane jtt, al. (14)
have parti ally purified the phytochrome pigment.
Visible radiation with selected wavelengths also has an
effect on i nternode elongation of intact plants. Some workers
have suggested that the absence of short wavelength light
produces an Increase in stem length (38). Meij er (18) re-
ported that Calendula and i'etunia plants exhibited a pro-
nounced elongation of in ternodes in blue light or in darkness.
However, other plants such as Mlrabili s. fiivi na. and Mentlm
showed a pronounced i nternode elongation in red or green
light and had short internodes in blue light.
Using relatively low intensity radiations (about 500
ergs 'cm- per second), Klein (13) subjected tissue cultures of
rarthenoci ssus crown-gall tissue to raonochromati c light in
the visible blue, green, orange, red, and far-red regions of
the spectrum. In Klein's study, green radiation repressed
growth, as measured by fresh weight, while all other wave-
lengths used produced growth of about the same degree above
the dark control. At somewhat higher intensities (5,000 ergs/
cm^ per second), green repressed growth even more. The growth
of the Parthenocissus crown-gall tissue also was inhibited by
full-spectrum radiation at about 1340 lux.
5
I n 1 9 5 7 , i t was demo nstrated t h a t phytochrome was n o t
t h e o n l y photoreac.tive system i n photomorphogenesis ( 3 0 ) .
The experimental d a t a c o l l e c t e d a t t h a t t i m e i n d i c a t e d t h a t
a d i f f e r e n t photoreactive s y s t e m was i n v o l v e d . T h i s r e a c t i o n
was demonstrated o n l y i f t h e m a t e r i a l was i r r a d i a t e d w i t h
r e l a t i v e l y h i g h i n t e n s i t i e s of r a d i a t i o n Cover 2000 e r g s / c m ^
p e r s e c o n d ) and h a s b e e n t e r m e d the "high-energy r e a c t i o n of
photomorphogenesis." The h i g h - e n e r g y reaction seems to be
more important than the phytochrome system under natural
conditions of high irradiance. A c c o r d i n g to Mohr ( 1 9 ) , the
system i s not photoreversible and the a c t i o n spectrum of the
reaction shows peaks i n the blue and far-red w i t h slight
activity i n the red. It was suggested t h a t a raetal-flavo-
protei n enzyme might be the light-activated species i n v o l v e d
i n the system.
I n v i e w of t h e l a c k of d a t a c o n c e r n i n g t h e r e s p o n s e s
of p l a n t t i s s u e c u l t u r e s t o h i g h i n t e n s i t y monochromati c
r a d i a t i o n s , and w i t h t h e c o n v i c t i o n t h a t i n vitro cultures
may provide a useful tool for s t u d y of the photoreactive
systems involved, the present investigation was undertaken.
I t was the purpose of the i n v e s t i g a t i o n to d e t e r m i n e the
effects of selected high i n t e n s i t y monochromatic r a d i a t i o n s
on the growth of pith callus tissue of P e l a r a o n i u B t zonale.
variety Enchantress F i a t . In addition, the extent of cell
differentiation was to be d e t e r m i n e d for t i s s u e s grown under
each experimental treatment.
CHAPTER BIBLIOGRAPHY
1. Bergman, Ludwig, "The Effect of Ki neti n on the Metabolism of Plant Tissue Cultures," Proceed!nas of the Inter-national fioafoyegge SJL PUlt, tissue Culture. California, McCutchan Publishing Company, 1963.
2. Butcher , D. N, and H. C. Street, "Excised Root Culture," Botanical Review. XXX (1964), 513-586.
3. D'Amato, Francesco, "Endopolyploidy as a Factor in Plant Tissue Development," ffgg &£ Int Conference on Plant Tissue Culture. California, McCutchan Publishing Company, 1963.
4. De Capite, Luigi, "Action of Light and Temperature on the Growth of Plant Tissue Cultures I j Vitro American Journal of Botany. XLII (1955) , 669-873.
5. Gautheret, R. J., "Action de la Lumifere et de la Temper-ature su la N^oforaiation de Racines par de Tissus de Topi nambour Cultiv£s jLa Vitro Comptes Rendus Acadgmi e Pes Sciences. CCLII (1961) , 2791-2796.
6. . "Culture du Ti ssu Cambial," Comptes Rendus Acad^ttle Pes Sciences. CXCVIII (1934), 2195-2196.
7. "La Culture des Tissus V£g£taux. Son Histoire, ses T e n d a n c e s R e v u e Cvtoloaie Bioloqie V^adtales. XXVII (1964), 99-200.
8. . "fiecherches sur le Bourgeonnement du Tissu Cambi al d'Ulmus campestris Cultivi Ij, Vitro." Comptes Rendus Acadgmie Des Sciences. CCX (1940), 632-634,
9. "The Nutrition of Plant Tissue Cultures Annual Review of Plant Physiology. VI (1955), 433-484.
10. Heller, Ren£, "Some Aspects of the Inorganic Nutrition of Plant Tissue Cultures," Ml Utt. International Conference on Plant Tissue Culture. California, McCutchan Publishing Company, 1963.
11. Hildebrandt, A. C., A. J. Ri ker, and B. M. Duggar, "Growth Xj, Vitro of Excised Tobaeco and Sunflower Tissue With Different Temperatures, Hydrogen-ion Concentrations, and Amounts of Sugar," American Journal of Botany. XXXII (1945), 357-361.
«
12. h i l i i e b r ^ a u t , <*>. C. H i k e r , aitu B, 61. Uuygar , "Tito l u f luence of t l i o Compos i t ioa of the Keuiuia on tb« Or out Ik l a V i t r o , o f Kxeisea Tobaeco and Sunf lower T i s s u e C u l t u r e U 9 4 6 ) , 591-G97.
a l . B.o.t.M.a.v.. aAaIII
13. K l e i n 1. M . , "Rep ress i on o f T i s s u e C u l t u r e Growth by V i s i b l e «tnd Near V i s i b l e R a d i a t i o n , " P l a n t AXXI A. ( 1 9 6 4 ) , 536-539.
14, Lane , H. t . , i t . tf. Si®oelatao? C. M. F i r e * , a ad V L . B u t l e r , " I 'bytochrotao froia Creen P l a n t s ,n tMaai.
AAAVI I I ( i 9 6 0 ) , 414-416.
15,
16-
H I
Rue, t . U . , " r e g e n e r a t i o n i n M u t i l a t e d Seed l i ngs tE&LSSiULmS. E l ( 1 9 3 3 ) , 53-67
LSf#re6ttt . J . L
. t ialMiMul
»od J . Uonner, "The I o t e r a c t i o n of
AlA
"'I *> to A t
Aux in and L i y h t l a t he Growth fiesponses o f P l a n t s &£ 4te-
C1953). 905-916. 4c..ttcftffiy. £ l SfijUftSSB > AXAIA
17. Lockha r t J . a . , " t h o t o i n h i b i t i o n o f Steia h l o n g a t i o n by F u l l So la r l i a t l i u t i o n A L V I I I ( 1 9 6 1 ) , 3 6 7 - 3 % .
Journa l . aX a.t.a..«LY
16). S l e i j e r , *i., " f -hctoaorphegesie i i is i n D i f f e r e n t S p e c t r a l * * " - - - - - — a, i a Regions jattii. Efij
e d i t t n i by R ^1 throw . AIMi ririiniiireimiiTTH-T- - - » '?»aaUiagtou, ft. C . , American A s s o c i u t i o n f o r the ndvancecieut o f Sc i ence, 1959.
19. a o h r , I I . , MTho C o n t r o l o f r i a n t Growth ««m fc©vi»loptaent by l / i gh t A *-
,v!a (1964) , c 7 ~ l i :
Nae f , J . , " A c t i o n de l a Luiai&re sur 1 * « t i l i z a t i o n du Glucose par l e s T i s s u s Vt fg l taux C u l t i v t f s l a V-
M a a r n .< 1706-1706.
Narayanstfaft i t S, and h. ^ o r s t o g , " i ' l a n t Kiabryo C u l t u r e S o v i e t a A A (1964-1 , 507-620,
N i c k e l l , L . ( i, 41)0 U. .S. d u r k h o l u o r , " n t y p i c a l Growth o f i' l a n t s . I I . Growth i a V i t r o o f V i r u s T imor# o f Kmaex i n R e l a t i o n to Tewpera t i i r a , pit ano Va r i ous Sources o f N i t r o g e n , Carbon and S u l f u . *Macrl.c..a,n SlL Dotaav . AAAV' 11 (1950) , 53{.''—G4 < .
23. Nitsch, J. P. , "Growth and Development In Vitro of Ovaries," American Journal of Botany. XXXVIII 566-577.
24.
*3 n Urn *J •
Exci sed (1951),
Pollard, Growth-of the (1959),
J . K . , E. I promoting Activity of Non-ionic Components, Supplement, vii.
Shantz, and F. C. Steward, "The Coconut Milk: the Nature ' Pi ant UMialfilaax? ^xxiv
Rappuport, J., "In Vitro Culture of Controlling Their Growth,' Factors
XX (19C j4)
Plant Embryos and Botanical Review.
16.
27 *
Hiker, A. J. and A. C. Hildebrandt, "Plant tures Open a Botanical Frontier," Annual Microbiology. XII (1958), 469-490.
Tissue Cul-
flevlfiw al
Robbing, W. J., "Cultivation of Excise* Stem Tips Under Sterile Conditions," LXXIII (1922), 376-390,
! Root Tips and Botanical Gazette,
28.
29.
Eobbins, W. J. on Growth of
and W. E, Maneval, "Further Experiments Excised Root Tips Under Sterile Condi-
tions," Botanical Gazette. LXXVI (1923), 274-287
and F. C. Steward, "The Identification ft
of Shantz, E. M Compound A from Coconut Milk as 1,3-Diphenylurea, Journal of the American Chemical Society. LXXVII (1955) 63S1-6353.
30. Siegelman, H. K. and S. B. Hendricks, "Photocontro1 of Anthocyanin Formation in Turnip and Bed Cabbage Seedlings." Plant Phvsioloov. XXXII (1957), 393-398.
31. Skoog, F. and C. 0. Miller, "Chemical Control of Growth and Organ Formation in Plant Tissues Cultured Jjj. V i t r o B i o l o g i c a l Action of Growth Substances. Symposium of the Societv for Experlmental Biology. XI (1957), 118-131.
32. Skoog, F. and C. Tsi, "Chemical Control of Growth and Bud Formation in Tobacco Stem Segments and Callus Cultured In (1948) , 782-
Vitro . •787.
American Journal of Bo.ta.nv. XXV
33. Stei nhart, LZ Vitro
S. and Growth
F. Of
Skoog, "Nutrient Requirements for
Botanv. XLVIII (1961) Spruce Tissue," Aaeric.an Journal &£
•472.
34. Stei nhart, S., L. Anderson, and F. Skoog, "Growth-promoting Effects of
Pto*9,l9ffV
Cyclitols on Spruce Tissue Cultures," Plant. XXXVII (1962), 60-66.
9
35. Steward, F. C. and S. M. Caplin, "A Tissue Culture from Potato Tuber: the Synergistic Action of 2,4-D and Coconut Milk/' Science. CXIII (1951), 518-520.
Street, H, E. Excised Soot Culture," Bioloqjcal Review XXXII (1957), 117-155. ** ** 4
36
37. Tulecke, 'V. , "liecent Progress and the Goals of Plant Culture," B a ^ e y j the Torrev Botanical CIA.
LXXXVI (1961) , 283-209.
38. aiassink, E. C. and J. A. J. Stolwijk, "Effects of Light Quality on Plant Growth," Review Plant Physiology. ¥11 (1956), 373-400.
39. White, P. R., a Plant Tissue Culture. Lancaster, Pennsylvania, Jacques Cattell Press, 1943,
4 0 • — "Nutritional Requirements of Isolated Plant
™ S ? ? a l , r d o o t 9 o ! ! * , M A ™ a J L Sll U a H l Phvsioloov, II (1951), 231-244.
• • i > "Plant Tissue Cultures," Botanical Review. II (1936), 418-437. " '
42 - "Plant Tissue Cultures." Bioloorical Revi XII (1941), 521-529. " ~J^Lk
ew.
43- — "Potentially Unlimited Growth of Excised Tomato Root Tips in a Liquid Medium," Plant Physiology. IX (1934), 585-600.
44. , "Potentially Unlimited Growth of Excised Plant Callus in an Artificial Nutrient," «*merican Journal of Botany. XXVI (1939), 59-64.
45. — ^ I M Cultivation slL M L & k l I M Plant Cells. New York, Ronald Press, 1963.
46. A'hite, P. R, .and P. G. Ri ss er, "Some Basic Parameters in the Cultivation of Spruce Tissue," Phvsiolaia Plantarum. XVII (1964), 60U-619.
CHAPTER I I
MATERIALS AND METHODS
Initial callus cultures were obtained from pith tissue
of greenhouse-grown Felaraonium zonale variety Enchantress
Fiat. The plants were grown from cuttings and were about
ten months old at the time of harvest. Stock plants were
obtained from Jack Cannon Florist, Arlington*, Texas.
Procedures similar to those presented by Chen and
Galston (1) were used to initiate pith callus growth. Stem
segments, three to four centimeters long, were taken tea
centimeters below the apex of the stem. After removing all
leaves and bu<; s, each segment was washed with tap water,
sterilized in a beaker containing 15 per cent commercial
Clorox for thirty minutes, and rinsed three times with
sterile, distille<i, deraineralizee! water. The pith was cut
into small, uniform disks by first boring cylinders of tissue
with a sharpened glass tube and then slicing disks of uniform
thickness with a glass knife. The resulting disks were 6.0
millimeters in diameter, 1.0 millimeter thick, anil weighed
about 20 milligrams, fresh weight. The ciisks were then
inoculated onto a solid medium contained i n twenty-five by
one hundred millimeter culture tubes, two disks per tube.
10
11
The tubes were capped with loose-fitting aluminum caps that
permitted aeration and prevented contamination from the
atmosphere.
The medium used was White's basic inorganic (4), sup-
plemented with organic constituents (1), and solidified with
one per cent Difco Io n-Agar. Table I lists both the inorganic
and organic constituents of the modified medium used.
TABLE I
COMPOSITION OF WHITE'S MODIFIED MEDIUM
Constituents Milligrams Per Liter
Inorganic;
KCL KNOg ca(No 3)o*4H 26 MgS04.7fl2° • • • NapSO^ . . . » NaHvP04 * H 20 Fe2^S04)g . . . . . . . MnS04-41120 ZnS04*7H20 . H3BO3 KI CuSC>4-5H20 MoOg
Organic: Thiamin , . . Pyri doxi ne Nicotinic Acid Glycine . . . mvo-Inositol
Kinetin (6~furfury1 aminopuri ne) 2,4-dichlorophenoxyacetic acid
65 0 80 0
300 0 720 0 200 0 16 5
e %}
7 0 3 0 1 5 0 75 0 001 0 0001
0 1 0 5 0 5 2 0
100 0 >000 0
0 1 0 1
All solutions were made in distilled, demi nerali zed
water. The pH of the medium was adjusted to 5.3 with hydro-
chloric acid or potassium hydroxide before addition of the
agar. After heating a no addition of the agar, the medium was
dispensed to the culture tubes, twenty milliliters per tube,
and autoclaved at 1.2 kilograms per square centimeter pres-
sure for fifteen minutes at 121 degrees Celsius (C).
The preparation of pith explants and all subsequent
operations involving the tissues were performed in a stain-
less steel sterile transfer chamber, the inside of which was
washed with 10 per cent commercial Clorox and then irradiated
with ultraviolet light for at least four hours prior to use.
The pith explants were incubated in a dark chamber at
27+2° C. After three weeks of culture, approximately 60 per
cent of the explants had developed callus tissue ranging
froia one- to three-fold over the initial volume. Callus
pieces from these initial cultures were used as the inocula
for subsequent procedures (see Figure 8, I3-C).
Pieces of pith callus from the initial cultures were
excised, weighed individually to the nearest milligram on a
Mettler Model H16 analytical balance, and placed in culture
tubes. Weights were made by placing the tissue in a sterile
petri dish, weighing, and taring the dish after the tissue
had been removed, A sterile filter paper inside the petri
dish served to remove excess water from the tissue. All
weights were made inside the sterile chamber. Three pieces
of tissue, ranging in weight from eighteen to thirty milli-
grams, were placed in each tube. All manipulations were made
in diffuse, incandescent light. The use of colored "safe"
13
light was avoided since experimental treatments consisted of
monochromatic light within the sarne wavelength range. The
extreme friability of the callus tissue made it possible to
use glass loops fashioned from glass rods for excising and
transferring the tissue.
Three tubes, each containing three callus pieces selected
from three different stock tissues, were placed in each of
five culture chambers to receive the respective light or dark
treatment. Figure 1 illustrates the apparatus used to culture
and irradiate the callus tissue.
Light treatments consisted of monochroraati c radiation
obtained by filtering incandescent light from General Electric
Reflector Flood lamps through layered, plexiglass filters (2).
A relatively full spectrum treatment ("white") also was admin-
istered with a General Electric Lumiline incandescent tube
by passing the light through transparent plexiglass. Three
one hundred-watt lamps were used for the red and green treat-
ments , and a one hundred fifty-watt lamp used for the blue
treatment. Variations in intensities were obtained by adjust-
ing the height of the lamps over the filters. A Fowerstat
variable transformer was used to adjust the radiant output of
the light sources to 15,000 ergs/era2 per second as measured
with a YSI-Kettering Model 65 Radiometer. • The radiant energy
measurements were made at the level of the culture tubes.
Three monochromatic treatments were used: one in the visible
blue, the filter system transmitting wavelengths from 410
14
millimicrons to 480 millimicrons and peaking at 450 mi 1 li-
micronsj one in the green, the fiIter system t r a n s m i t t i n g
from 500 millimicrons to 590 millimicrons and peaking at 545
millimicronsj and one in the red, the filter system trans-
mitting from 600 millimicrons to 690 millimicrons and peaking
at 650 millimicrons. The transmission data were obtained
from published spectral transmission curves for the filters
used (2). All light treatments were continuous. The dark
treatment was accomplished by use of a light-tight chamber
heated with a thermostatically controlled hot plate (see
Figure 1).
Temperatures were m a i n t a i n e d at 2 7 + 2 ° C. by an oxygen-
tent coo l i n g unit. Figure 1 i l l u s t r a t e s the manner in which
the unit was attached to the treatment chambers.
After twenty-two days of culture, each tissue piece was
individually weighed (fresh weight). Microscopic examina-
tions were made to d e t e r m i n e the extent of differentiation.
Since lignification is often an indication of differentiation,
at least 1n vessel elements (3), phloroglucino1-HC1 stain was
used to aid In determining the nature and degree of differ-
entiation of the callus tissue. Di r e c t observation of cells
was made by preparing fresh mounts of different parts of
representative tissues. No microtome sections were prepared
because the callus tissues were extremely friable and were
easily broken into f r a c t i o n s s u i t a b l e for wet mount preparu-
tions. Photographs of the callus tissues w e r e made with a
15
Polaroid Land camera equipped with a close-up lens. Photo-
micrographs of fresh mounts of callus tissue cells were made
with an AO Spencer Series 10 Microscope equipped with a
35 millimeter camera.
This investigation was designed to permit a statistical
analysis of the data, using a randomized b l o c k design and
the s t u d e n t ! z e d range test. All experiments were repeated
three times. For each test, new media were prepared and
precautions were taken to obtain identical conditions. The
first test was begun on March 23, 1966, and the last o n e ended
on J u n e 12, 1 9 6 6 .
16
Figure 1--Uiayram of the tissue culture chamber. A, Light source. B, Filter. Cf Conduit carrying cool air to rear of chambers. D, Culture tubes. E, Conduit. F, Oxygen-tent cooling unit. G, Hot plate supplying heat to dark chamber. H, Entry of cool air at rear of chamber. I, Intercompartmental air port, culture shelves.
J. Dark
CHAPTER BIBLIOGRAPHY
1. Chen, H. and A. W, Gulston, "Growth and Development of Pelargonium Pith Cells Ij» Vitro Phvgloloala P1antarura. XVIII (1965), 454-461.
2. Klein, R. "The Hole of Monochromatic Light in the Development of Plants and Animals," Carolina Tips. XXVII (1964), 25-26.
3. Lipetz, J., "Mineral Elements and Differentiation in Plant Tissue Cultures," Proceedi nas of the International Conferenee on Plant Tissue Culture. California, McCutchan Publishing Company, 1963.
4. White, P. B. , TJia (^Itl.v^fqn Anjj&i M & Plant Ceils . New York, Ronald Press, 1963.
17
CHAPTER III
RESULTS
A f t e r twenty-two days of culture, all tissue u n i t s were
pale yellow in color and were quite friable. Maximum growth
resulted in fresh weight increases representing a twelve-fold
increase over the initial weight. U in imum growth was repre-
sented by tissue units which did not show a fresh weight
increase over the initial weight. The initial and final
fresh weights of each piece of callus tissue used in this
experiment are presented in Table II. That the growth of
the tissue is quite variable with respect t o fresh weight
increases is apparent in the variability of final weights.
The mean weight increases shown in Table II reveal that
greatest growth occurred In the blue and " w . h i t e " light treat-
ments and that less growth occurred in the dark treatment
than in a n y o t h e r t r e a t m e n t .
All three trials yielded similar results w i t h respect
to treatments producing maximum and mi nimum growth. However,
the mean increases for the red and green treatments, respec-
tively, in trial one w e r e reversed in trial two and three.
The mean for each t r e a t m e n t of the combined trials is pre-
sented in Figure 2.
IB
19
TABLE I I
I N I T I A L AND FINAL WEIGHTS OF CALLUS T I S S U E OF PELARGONIUM ZONALE VARIETY ENCHANTRESS FIAT GROWN IN FIVE
DIFFERENT LIGHT TREATMENTS
F r e s h Weight I n M i l l i g r a m s
B lue Green Eed Dark " W h i t e " T r i a 1 (-45.0 MU) ( 5 4 5 mu) (6£ >0 mu) JL £ JL « X
and JN-«| ¥*Mf <** 1*4 T i s s u e m
«|M| m
•$** r*4
m
***4 « «i"4
**•4 m
fMf Number ** m +* 03 03 m Number £ •HI St S3 S3 *£*4 a
S3 S3 S3 a •H 53 •HI
M HI W HHI Ut
A. 1 21 1 2 7 18 3 0 20 75 27 27 21 175 2 1 8 27 28 65 2 0 92 25 97 18 60 3 27 95 2 9 1 5 5 2 6 50 19 4 0 18 125 4 19 6 0 19 1 9 0 23 121 25 55 21 21 5 19 1 0 0 19 4 7 19 19 21 121 29 81 6 25 8 0 10 4 0 19 26 19 2 0 22 147 7 23 1 1 0 25 2 5 18 18 24 81 19 84 6 2 4 141 28 79 21 107 22 24 26 166 9 22 134 25 9 0 z§ „ 77 , 25 . ,-25 .27. ... 27
MEAN .22 97
CM 80 2 1 6.5 23 5.3 .-2.2 .. .96
S . B . 2 ; a ' 4®. 9 4 . 4 5 4 . 6 2 , 5 -36.. 9.-] -.2urI. ...3.4 *.8 3..7... . 54.. 5
S . I 18 1 3 6 18 5 5 2 0 90 2 8 118 3 0 9 0 2 18 1 6 5 2 0 1 5 0 25 175 18 18 2 0 160 3 22 105 3 0 162 20 127 25 115 2 5 240 4 20 165 3 0 122 25 7 3 19 77 20 32 5 22 1 4 5 23 47 18 98 19 54 2 4 100 6 20 4 0 20 20 23 • « * 30 37 20 98 7 18 48 26 28 20 20 20 33 22 51 8 30 75 25 27 18 19 25 25 18 31 9 20 6.5........ 27 6.7 3 0 10.4 20 36
l e a n 20 99 24 .7.4 22 ,2 4 65 22 .93 S . D . .. ..4,2 5 3 . 6 „ 4 , 4 -5.2....6... 4 8 , 8 4 . 6 ...3.7, 6... 3 . 5 ...65.4
C, 1 19 211 1 8 2 0 20 18 19 5 0 18 8 3 2 17 55 22 5 0 20 73 18 109 29 127 3 2 8 6 0 24 * # # 20 82 21 68 24 117 4 25 111 23 3 0 22 45 19 22 21 52 5 20 22 21 92 25 106 20 56 22 98 6 22 2 5 19 216 26 29 22 1 8 16 ' 147 7 29 * * * 2 0 81 18 55 22 26 28 19 8 21 mm# 21 7 5 20 202 2 4 43 22 47 9 M , . f( .f. . 23 A O ! 18 . 21 „ 1 8 .. 7 2 19 . 3 1 .
Mean 22 6.4 21 KF, *J O 21 7 1 20,,, §2 22 8 0
......1,0.:. 4 . 2 J i £ A L . A - 9 , ^ T 5 2 . 9 54 F 5 1 . 9 jyLdL. 3 . 8 4 2 . 7
20
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21
Photographs of the final tissues from each treatment in
trial two are presented i si Figure 3, A (1 -5) . Although
volume differences are not so apparent in the two~dimen$i onal
photographs, the fresh weight differences given in Table II
reveal the growth differences of individual tissue units in
each treatment. The smallest piece of tissue shown in Figure
3, A C D , represents the relative size of the initial tissue
units and the largest piece in the same photograph represents
the maximum size attained by any single tissue unit.
Photomicrographs of typical cells from representative
callus tissues are shown In Figure 3, D~H. The character-
istically large, highly vacuolated cells found in all tissues
are shown in Figure 3, D. These cells ranged in shape fro®
nearly spherical to greatly elongated cells. The spherical
cells were 0.05 to 0.1 millimeter in diameter whereas the
elongated cells were about 0.1 millimeter in diameter and up
to 1.0 millimeter in length. The cells shown in Figure 3, 0
were magnified 100 times. The individual ceil shown in
Figure 3, E was magnified 450 times and was included to show
the eellular inclusions of a typical callus cell.
All tissues yielded callus growth under each treatment
and no shoot or root formation was observed. However, the
tissue did develop aggregates of differentiated material in
the form of vessel elements. Figure 3, F is a photomicro-
graph of a typical aggregate of vessel element cells found
22
f I « •* % n • I *' * I »F •
V,'."'
* 4 #
* 0 *
n % *
*
'. %"* "**
V r-v ' • » ' * ' * •
.
: "
Figure 3—Photomicrographs of Callus Tissue of Pelargonium aonale. variety Enchantress Fiat. a, Sets of tissue from each light treatment in trial two. Numbers 1-5 represent blue (450 mu) , green (545 mu), red (650 mu), "white," and dark treatments, respectively. Two-thirds actual sizes are shown. B, Disk of telaraoni urn pith tissue before cultivation, X2. C, Pith tissue from initial explant after three weeks of culture, X2. 0, Typical callus tissue cells, XI0 E, Single callus tissue cell, X450. F, Aggregate of differentiated cells, X10Q. G, Aggregate broken apart to show individual elements, X100. H, Individual vessel element, X450.
scattered throughout all tissues. A typical aggregate, spread
apart to reveal individual elements, is shown in Figure 3, G.
A higher magnification (X450) of a typical vessel element
common in tissues from all treatments is depicted in Figure
3, II. The secondary thickening of the cell wall is distin-
guishable. It was not possible to determine a significant
difference in the degree of cell differentiation between
treatments. Phloroglucinol-HCl staining revealed a high
degree of lignin deposition in the aggregates and in indi-
vidual vessel elements.
A statistical analysis using mean fresh weight increases
for each treatment with a randomized block design revealed
significant differences at the one per cent level of s i g -
n i f i c a n c e due to treatments (Table III).
24
TABLE I I I
VARIANCE TABLE FOR DIFFERENT LIGHT TREATMENTS ON MEAN FRESH WEIGHT INCREASES OF CULTURES OF PELARGONIBM ZONALE GROWN
IN VITRO AS DETERMINED BY USING A RANDOMIZED BLOCK DESIGN FOR ANALYSIS OF VARIANCE
Treatments—Mean Fresh Weight Increase in Milligrams
Trial Blue Green Red Dark "White"
A 75 58 44 3 0 76
B 79 50 62 41 7 1
C 6 2 3 4 50 32 58
Source of Variation SS DF MS F
Treatments 2975.0 4 7 4 4 . 0 1 4 . 8 8 * ®
Blocks 4 6 3 . 6 2 231.G 4 . 6 0 ®
Error 4 0 2 . 4 8 50.3
Total 3 7 4 0 . 8 14
*0.05 level of significance. **0.01 level of significance.
Significant differences between individual treatments
were determined with the student!zed range test. Results of
the test, presented in Table IV, showed significant differ-
ences at the five per cent level between blue and red, blue
and green, blue and dark, "white" and red, "white" and green,
"white and dark, and red and dark.
25
TABLE IV
SIGNIFICANT DIFFERENCES OF MEAN FRESH WEIGHT INCREASES OF CALLUS TISSUE OF PELARGONIUM ZONALE BETWEEN
INDIVIDUAL LIGHT TREATMENTS AS DETERMINED BY THE STUDENTIZED RANGE TEST
T r e a t m e n t Dark Green (545 rau)
Red (650 mu)
"White"
Blue (450 mu) 36® 25* 20* 4
" W h i t e " 34® 21* 16*
Red 18® 5
Green 13
* 0 , 0 5 level of s i g n i f i c a n c e ,
CHAPTER XV
DISCUSSION
The growth of the Pelargonium pith callus tissue was
significantly greater in the blue and full spectrum ("white")
treatments than in the red, green, or dark treatments. In
the blue and full spectrum treatments, the d i f f e r e n c e s in
weight i n c r e a s e s were not significantly different« Thus the
action spectrum of the Pelaraonium callus tissue growth is
comparable to the high-energy action spectra reported for
several plant systems, including s y n t h e s i s of anthocyanin
(8, 9), s y n t h e s i s of flavonols (9), control of hypocotyl
lengthening (8), and internode elongation (2).
The action spectrum of the high-energy r e a c t i o n in the
range of the visible spectrum has been determined for growth
phenomena of several plant species. It shews peak effective-
ness in the far-red and in the blue portion of the visible
spectrum (7, 8, 9).
Wassink (10), using radiant intensities of
10,000 ergs/cor per second, r e p o r t e d that stem elongation in
Hvoscvamus niaer was more pronounced under blue and far-red
irradiation than under green, yellow, or red. The a d d i t i o n
of red to blue inhibited the promotive effects of blue. The
addition of red to far-red treatments produced some inhibition
26
27
but less than in the blue light. It was proposed by the
authors (Wassinlc jtJL.) that far-red and blue have similar
effects on stem elongation of Hvoscvamus and that red light
suppresses elongation. Green light was shown to have no
positive effects on stem elongation. Similar results were
obtained by Fletcher a 1 • (2), who irradiated bean seedlings
with relatively high intensities of monochromatic blue, red,
and far-red and observed greater elongation under the blue and
far-red treatments along with an apparent suppressive effect
of red light. The same workers also showed that root forma-
tion of the bean plants was controlled by the blue/far-red
system in that both wavelengths i nhi bi ted formation to the
same degree.
Since the full spectrum source ("white") used in the
present study to irradiate the callus tissue of Pelargonium
was an incandescent lamp, and since this source emitted far-red
radiation, it is possible that the growth effects observed
under this treatment were due to the far-red light. However,
it should be remembered that the full spectrum treatment also
contained blue, red, and green wavelengths and that no data
are available concerning the effects of light combinations on
callus tissue growth. The full spectrum treatment effects
were consistent with the reported actions of blue and far-red
irradiation under relatively high intensities of light and
would well explain the low degree of growth response that
occurred in the dark treatment relative to the full spectrum
treatment.
28
In their work with dodder s e e d l i n g s , L a n e and K a s p e r b a u e r
(6) separated two photoresponges associated with hypocotyl
hook o p e n i n g and stern t w i n i n g . Hook o p e n i n g was dependent on
the low-energy red/far-red system w h i l e t w i n i n g action w a s
controlled by high-energy blue and far-red irradiation. The
nature of the results prompted these authors to propose two
different photoreceptors involved in the high-energy reaction,
one sensitive to blue and the ether, perhaps phytochrome
itself, sensitive to far-red. According to Mohr (T) and
Vinee (9), the high-energy system is not photoreversible and
may operate simultaneously with the low-energy phytochrome
s y s t e m , e i t h e r synergi stically or independently. In either
case the high-energy reaction wi11 override the low-energy
system. It is, of course, possible that phytochrome was
present in the Felaraoniurn callus tissue and that a low-
energy reaction was o p e r a t i n g along with the proposed high-
energy reaction. However, there have been no reports on the
presence of phytochrome in plant callus tissue and the only
action s p e c t r u m reported for the growth of such t i s s u e at
low energy levels was not the action spectrum of the phyto-
chrome systern (5).
The theoretical background of the high-energy reaction
has not yet been firmly e s t a b l i s h e d . Mohr (8) proposed the
i n v o l v e m e n t of metal-flavoproteins, such as butyryl-coenzyme A
dehydrogenase, which have absorption spectra s i m i l a r to the
action spectra of the high-energy reaction.
29
It is possible that the effects of the blue and full
spectrum light treatments on the growth of the Pelargonium
callus ti s s u e were brought about by the induction of changes
in the levels of certain enzymes, flavonoids, and/or related
phenolic compounds. Indoleacetic acid oxidase may be regu-
lated by flavonoids in isolated systems. Some flavonoids
inhibit indoleacetic acid oxidation w h i l e others enhance it
(6). The action spectrum for flavonoid b i o s y n t h e s i s (8) is
similar to the one presented in this paper for the growth of
Felaroonium callus tissue.
In addition to enzyme effects, total pr o t e i n synthesis
has been shown to be dependent on light. tucker (11) reported
that p r o t e i n synthesis was dependent on light in isolated
disks of potato tuber tissue. The action spectrum for the
response showed a broad maximum in blue light.
That the Pelargonium callus tiss u e did not develop dif-
ferentiated tissue beyond simple vessel elements is consistent
with results reported by other workers. Chen and Galston (1),
the only workers who have reported the use of Pelargoniura
tissue cultures, stated that only tracheids were formed in
the pith callus tissue. There might be some question as to
the sole presence of tracheids in angiosperm tissue. It is
more probable that the structures observed by Chen and Galston
were vessel elements rather than tracheids.
The fin d i n g that no differences occurred in the degree
of d i f f e r e n t ! a t ion under the different light and dark treatments
30
appears to be consistent with the findings of Gautheret (3)
that wavelength is not important in differentiation when high
i n t e n s i t y light is used.
Although the growth factors, ki neti n and 2,4-dichloro-
phenoxyacetic acid, used in the Pelaraonium culture media
have been reported to i n i t i a t e bud and root formation, respec-
tively, it has been reported that balanced a m o u n t s of kinetin
and inaoleacetic acid produce n e i t h e r bud nor root formation
(4). Since the action of 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid is
s i m i l a r to that of i ndoleacetic acid, it is proposed that the
0.1 milligram per li ter concentrations of both kineti n and
2,4-di chlorophenoxyaceti c acid used in the Pelargonium culture
m e d i a c o n t r i b u t e d to the lack of organ formation in all treat-
ments .
The findings of this investigation Indicate that the
high-energy reaction typical of intact plants is also in
o p e r a t i o n in pith c a l l u s tissue of Pelargonium zonale. variety
Enchantress F i a t , grown Jj, vitro under high i n t e n s i t y mono-
chromatic radiations. It can be inferred from these findings
that the tissue con t a i n s the photoreceptors) involved in the
high-energy reaction and that the factors necessary for the
response were synthesized in the jy| vitro cultures or were
transferred from the initial explants. Further work with the
rate of tissue growth and w i t h the degree of r e s p o n s e with
age of tissue would help to elucidate the nature of the
photoreceptor(s) involved.
31
In a d d i t i o n , t h e s e r e s u l t s , and t h e f i n d i n g s of o t h e r s
c i t e d i n t h i s p a p e r , i n d i c a t e t h a t t h e i n f l u e n c e s of l i g h t on
p l a n t t i s s u e c u l t u r e growth o u s t be t a k e n i n t o accoun t when
such c u l t u r e s a r e used f o r e x p e r i m e n t a l p u r p o s e s . I f p l a n t
t i s s u e used f o r experimental p u r p o s e s r e s p o n d s to e i t h e r t h e
low- or h i g h - e n e r g y reactions, and most plant t i s s u e s have
been shown to be sensitive to one or both, then the cultures
should be g r o w n i n either the dark or under specifically
d e f i n e d light regimes, preferably those for which growth
responses have been d e t e r m i n e d . For P e l a r g o n i u m zonale. the
use o f ful1 spectrum, high intensity i l l u m i n a t i o n for routine
o r experimental cultivation approximating natural conditions
i s i n d i c a t e d .
K e e p i n g in mind the v a r i a b i l i t y that usually is observed
i n callus t i s s u e g r o w t h , and the even more variable d a t a avail-
able concerning the effects of light on plant growth , i t i s
o b v i o u s that a g r e a t d e a l of work should be done in both
a r e a s . Improved c u l t u r e t e c h n i q u e s must be d e v e l o p e d t h a t
w i l l y i e l d r a p i d , c o n s i s t e n t , and u n i f o r m growth of j j i v i t r o
plant t i s s u e s before these cultures can be used w i t h a high
d e g r e e of c o n f i d e n c e i n experimental p r o c e d u r e s .
At p r e s e n t t h e f i e l d of photomorphogenesi s is i n an
a c t i v e s t a t e i n d e e d . As more work i s r e p o r t e d and more d a t a
a c c u m u l a t e d t h e rather elusive photoreactive s y s t ems i n v o l v e d
i n t h e b i o l o g i c a l r e s p o n s e s of p l a n t s to l i g h t w i l l p e r h a p s be
e l u c i d a t e d .
CHAPTER BIBLIOGRAPHY
1. Chen, B. and A. i. Galston, "Growth and Development of Pelargonium Pith Cells ia lilES.," Pfrml.fttolft i U M t i m i , XVIII (1965), 454-461.
2. Fletcher, B. A., R. L. Peterson, and S. Zalik, "Effect of Light Quality on Elongation, Adventitious Boot Pro-duction and the Relation of Cell Number and Cell Size to Bean Seedling Elongation," Plant Physiology. XL (1965), 541-548.
3. Gautheret, R., "Recherches sur 1 a Bourgeonnement du Tissu Carabial d'Ulmus c.aiap.e.s.tri.s. Cultivd Jja Vitro." C.o«jp_t.e.». Rendus Acadlmie Pes Sciences. CCX (1940), 632-634.
4. Hai ssig, B., Forest Tree 607-626.
"Organ Formation In Vitro as Applicable to Propagation," Botanical Review. XXXI (1965),
5. Klein, R. M,, "Repression of Tissue Culture Growth by Visible and Near Visible Radiation," Plant Physiology. XXXIX (1964), 536-539.
6. Lane, H, C. and M. J. Kasperbauer, "Photomorphogenic Responses of Dodder Seedlings," Plant Physiology. XL (1965), 109-116.
7. Mohr, H., "Primary Effects of Light on Plant Growth," Annual Review of Plant Physiology. XIII (1962), 465-488.
8. "The Control of Plant Growth and Development by Light," Biological Review. XXXIX (1964), 87-112.
9. Vince, D., "Photomorphogenesis in Plant Stems," Biological Review. XXXIX (1964), 506-533.
10. Kassi nk, E. C., P. J. A. L. De Lint, and J. Bensi nk, "Some Effects of High-Intensity Irradiation of Narrow Spectral Regions," Photoperiodlsm and Related. PhenoMena in Plants and Animals. Washington, D. C., American Association for the Advancement of Science, 1959.
11. Zucker, M,, "The Influence of Light on Synthesis of Protei n and of Chlorogenic Acid in Potato Tuber Tissue," Plant Physiology. XXXVIII (1963), 575-580.
32
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E l M l " " and the He1 Seedling Elongation, 548
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XXXIX
35
Lane, H. C. and M. J. Kasperbauer, "Photomorphogenie Responses of Dodder Seedling," Plant Physiology. XL (1965), 541-S48.
Lane, H. C., H, W. Siegelman, E, M. Firer, and W. L. Butler, "Phytochrorae from Green Plants," P1 a.nt Physiology. XXXVIII (1963), 414-416.
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Naef, J., "Action de la Lumi6re sur 1'utilization du Glucose par les Tissus V4g6taux Cultiv^s Iji V i t r o C o m o t e s Bendus Acad^nle Pes Sciences. CCXIIX (1959), 1706-1708.
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