In Vitro Regeneration and Protein Changes Associated With ...
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LSU Historical Dissertations and Theses Graduate School
1987
In Vitro Regeneration and Protein ChangesAssociated With Two Cultivars of Agrostis PalustrisHuds. Cultured Under High Sodium-ChlorideConditions.John Casper HovanesianLouisiana State University and Agricultural & Mechanical College
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Recommended CitationHovanesian, John Casper, "In Vitro Regeneration and Protein Changes Associated With Two Cultivars of Agrostis Palustris Huds.Cultured Under High Sodium-Chloride Conditions." (1987). LSU Historical Dissertations and Theses. 4361.https://digitalcommons.lsu.edu/gradschool_disstheses/4361
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I n v i t r o r e g e n e r a t i o n a n d p r o t e i n c h a n g e s a s s o c i a t e d w i t h tw o c u l t i v a r s o f Agrostts paluatris H u d s . c u l t u r e d u n d e r h ig h s o d i u m c h lo r id e c o n d i t i o n s
Hovanesian, John Casper, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1987
U M I.MX) N. Zeeb Rd.Ann Arbor, MI 48106
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IN VITRO R E GE NE RA TI ON AND P R O TEIN CHANGES A S S O C I A T E D WITH TWO CULT I VARS OF A q r o s t is p a 1 u s t r is H u d s .
CULTURED UNDER HIGH SO D I U M CHLORIDE CONDITIONS
A D 1 ssertation
Submitted to the Graduate Faculty of the Louisiana State U n i v ersity and
Agricultural and Mechanical College in partial fulfilIment of the
req u i r e m e n t s for the degree of Doctor of Philosophy
1 n
the Department of Hor t i c u l t u r e
by
John Casper H o v a n e s i a nBA, Univer s i t y of Connecticut, 1972BA, New E n g l a n d University, 1979
MS, University of S o u t h e r n Mississippi, 198 1May, 1987
ACKN O W L E D G M E N T S
I wish to thank the members of my committee, Drs. A l
bert C. Purvis, chairman, W i lliam J. Blackmon, James F. F o n
tenot, Ear 1 P. Barrios Jr., Donald W. Newsom, David H.
Picha, and Kenneth C. Torres for g u i d a n c e throughout the
various stages of this research. My a p p r e ciation is e x
tended to Ms. Rhonda Parche~ S o r b e t w h ose efficiency and
warmth has made the laboratory we spent so much time in a
pleasure, to Mr. Selim Cetiner with whom I participated in
many valuable academic and extra-curricular activities, and
to Mr. Paul C. St. Amand and Dr. Paul W. Wilson, who se wi 1 1 -
ingness to share their computer knowledge saved so many
hours. I would like to e x p ress my g r atitude to the D e p a r t
ment of H o r t i cu lt u re at Louisiana State University for
being a place which e n c o u r a g es close professional and p e r
sonal relationships. A special note of thanks to my very
good friend, James R. Ault, for thinking the way I do on
many i s s u e s .
My most heartfelt a p p r e c i a ti on goes to Dr. William M.
Randle, whose high professional and moral standards will
i i
always serve as g u i d elines in my career and in my personal
life. Without the many hours we spent preparing for the
next day's work, the q u a l i t y of life in Baton Rouge would
not have been the same.
There is always a person in one's life who provides the
warmth and lave that makes one feel special. It is to Ms.
Nancy Gordon Barker that I owe this debt. She has had a
profound and flattering influence in reshaping many of the
ways I now think and behave. Finally* to my mother,
Luciene* and to my father, A r s h a g ( who have always been
there with love and as s i s t a n c e throughout my life.
1 1 1
Table of Contents
Page
A c k n o w l e d g e m e n t s .................................................... i i
List of T a b l e s ....................................................... v
List of F i g u r e s ......................................................vi
Abs t r ac t v 1 i 1
I n t r o d u c t i o n ........................................................... 1
Chapter'
L i t e r a tu re R e v i e w ........................................ A
L i t e r a t u r e C i t e d ........................................ 17
I In Vitro Se l e c t i o n for Sodium ChlorideResistance* Regeneration, and Solution Culture E v a l u a t i o n of Two Cultivars of flqro s t 1 s palustr is H u d s ............................... 33
Abs tr ac t ..............................................33I n t r oduc t i o n ........................................ 35M aterials and M e t h o d s ............................ 36Resu I t s ............................................... A3D i s c u s s i o n ...........................................A 7L iterature C i t e d ................................... 51
II Syn t h e s i s of Salt Shock Proteins in CallusCultures of A q r o s 1 1 s p a l u s tris Huds. As a R e s p o n s e to So d i u m Chloride S t r e s s ................ 77
Abstrac t ..............................................77I n t r o d u c t i o n ........................................ 79M a t erials and M e t h o d s ............................ BOResu I t s ...............................................BAD i sc uss i o n ........................................... B5Literature C i t e d ................................... B6
Append l ............................................................... ^3
V i t a ................................................................... 10^
1 v
LIST DF TABLES
PageTab 1 e
Chapter 1 . In vitro selection -for sodium chloride resistance* regeneration, and solution c u lture e v a l u a t i o n s of two cul t i v a r s of A q r o s t 1 s palustr is Huds.
1 E f fects of 51A mM NaCl and generation upont r ansfer rable oalli of A_;_ palustr isPen n e a g l e ( ' P E ’ ) and Seaside ( 'S S ’ ) .......... .55
2 Effects of 51A mM NaCl on the p ro du c t i o n ofshoots and roots from callus cultures ofA . pal us t rls Pen n e a g l e < 'P E ' ) and Seaside( ' S S ’ ) on callus induc tion medlurn d u r i nydark i n c u b a t i o n ..........................................56
3 The effects of 51A mM NaCl on the percentageof cultures of A_ p a l u s t r i s Penneaq 1 e ( * PE ’ ) and Seaside ( ' S S ’) which had visiblyi nc r e a s e d ................................................... 57
A The effects of 51A mM NaCl an the perrentageof cultures of A_ palustr is P e n neagle <'P E ’ > and S e a side ( ' S S ’ ) with varying degrees o f nec r os i s ................................................ 5B
5 The effect of 51A mM NaCl on the productionof shoots and roots from callus c u l t u r e s of A . palustris P e nn ea gl e ( ' P E ’ > and Seaside( ' SS ’ ) on r eg en e r a t i o n m e d i u m ........................ 59
6 The effects of 51A mM NaCl on 1y o p h i 1 i zedweight and c o n c e n t r a t i o n of selected and nonselected callus cultures of A_. pa 1 u s t n s P en n e a g l e ( ' P E ’ ) and Seaside < 'S S ’ )................60
V
LIST OF FIGURES
F iqure
1
a
3
6
5
Page
Chapter 1. In vitro s e 1ec tlo n for sadlum c hloride resistance, regeneration, and s olution culture e v a l u a t i o n s of two cultivars of ftqros t i s p alustris Huds.
Visual rating chart for determining percent necrosis of pal us t r is' P e n n e a g l e ’ and ' S e a s i d e ’ calli cultured at 0 mM and 516 mM NaCl. From left to right: 0*/. ( + >, 1-35 (++), E 6 - 50 V. (+ + +),and greater than 51V. (+ + ++ )........................... 63
Respanse of A p al u s t r i s 'P e n n e a g 1e ’ and ’S e a s i d e ’ plants germin a t e d from caryopses ( ED > . pi an ts r egener a ted from calli cultured on O mM NaCl supplemented medium ( <^> ), and plants regenerated from c a l 1i selected from 516 mM NaCl supplemented medium c X ) , grown h y d r o p o n i c a l l y in 0 mM NaCl in H o a g l a n d ’s #3 nutrient m e d i u m ........................ 66
R esponse of A . p a l u s t r i s , ’P e n n e a q l e ’ and ’S e a s i d e ’ plants germinated from caryopses, cultured h yd ro po n i c a l l y in H o a g l a n d ’s HE nutrient med i u m at 0 mM NaCl ( ED > and at 516 mM NaCl ( )......................................... 66
R esponse of A . p a 1 us t r l s ■ ' P e n n e a q l e ’ and ' S e a s i d e ’ plants regenerated from calli cultured on O mM NaCl supplemented medium, grown h y d r o p o m c a l ly in H o a g l a n d ’s #3 nutrient medium at O mM ( ED 1 and at 516 mM ( \^> >N a C l ................. 6B
Response of A_;_ p a lust r i s , ' P e n n e a g l e ’ and ‘S e a s i d e ’ plants regen e r a t e d from calli selected from 516 mM NaCl supplemented medium, grown h y d r o p o n i c a l l y l n _ H o a g 1 a n d ’s #E nutrient me d i u m at 0 mM NaCl ( ED and and 516 mM NaCl ( )................................... VC)
Callus cultures from A . palustris ’P e n n e a g l e ’(t o p ) and 'Seas i d e ’ (bottom) cultured on a medium supplemented with 516 mM NaCl. Lighter areas are portions of cultures wher e certain cells are actively p r o l i f e r a t i n g .................... 73
v 1
P a g eF 1 qur e
7 R es p o n s e of A_^ pal us t r i s . ' Penneaq 1 e ' (topleft), and ' S e a s i d e ' (top right) plants re g enerated from N a C l - adapted c a l 1i , cultured h y dr op o n i c a l l y in H o a g l a n d ’s #2 nutrient medium at O mM NaCl- On the bottom are ft. palustr is plants regenerated from N a C l — adapted calli in 257 mM N a C l ............ 7ft
0 ftqrost t s palustr is, ' P e n n e a g l e ’ and' S e a s i d e ’ pla n t l e t s regenerated on medium supplemented with 51ft mM N a C l - Note succulence, dark coloration, and stunted morphology of many plants as a typical symptom of severe salt s t r e s s ................................... 7<b
Chapter 2. S y n thesis of salt shock proteins in callus cultures of ftqrostis p a l u s tris Huds. as a response to sodium chloride stress
1 One dimensional SDS p o 1y a c r y 1 amide gel slab depicting p r otein banding patterns of control and NaCl-adapted calli of ft_ p alustris ' P e n n e a g l e ’ and ' S e a s i d e ’ over threeg e n e r a t i o n s ...................................................93
v l l
Abstract: In vitro r e g e n e r a t i o n and p r o tein changes
associated with two cultivars of
Agrost i s palustr is Huds. cultured
under high sodium chloride conditions
Calli from ' P e n n e a g l e ’ and ' S e a s i d e ’ creeping bentgrass
(Aqr os t 1 s pa 1 us t r i s Huds. ) were selected for enhanced
ability to p r o l i f e ra te on media supplemented with 51A mM
NaCl. Based on dry matter produc t i o n and vigor, growth of
plants r e g enerated from NaC l - a d a p t e d calli of both cultivars
in saline hydroponic nutrient medium, was superior to that
of pl a n t s regenerated from non-adapted c a l 1i . At P57 mM
NaCl, plants from N a C l - s en si ti ve calli died or were unable
to p r o duce measur a b l e dry matter whereas plants from N a C 1 -
adapted calli grew well. Growth of plants from NaCl-adapted
calli was inhibited at 3A0 mM NaCl. At 51A mM NaCl, r e s i s
tant plants no longer produced measur a b l e dry matter, a l
though they remained alive.
The extra c t a b l e soluble protein content of NaC 1 -
adapted calli was less than that of n o n -adapted cultures.
E 1ectrophoretic banding patterns of soluble proteins from
N a C l - a da pt e d calli were compared with those from non-adapted
calli using one dimensional p o 1y a c r y 1 amlde gel e l e c t r o
phoresis. One particular band at 31.5fcd was unique to the
v i i i
N a C l - a da p t ed calli. The intensity of some bands increased!
while others decreased with increasing levels of N a C l -
adaptation. One g e ne ra ti on (30 days of dark incubation)
after being transferred to a medium without NaCl, p o l y p e p
tide banding patterns of NaCl-adapted calli were similar to
that of n o n-adapted calli.
INTRODUCTION
It has been predi c ted that there will soon be more
demand for food nearly e v e r y w h e r e in the world (20, 72, 73,
101). A c c ordlng to Tal (101), h o w e v e r , food production is
actually declining. The scarcity of arable land, the limits
of mod e r n technology, the high cost of energy, and decreases
in the funding of agricultural research have all contributed
to this downward trend ( EO » 72, 73, 101>. The opening of
marginally arable land to alleviate some anticipated food
shortages has, therefore, encouraged plant breeders to
produce cul t i v a r s which can tolerate environmental extremes
(20, 72, 73, 10 1).
Sodium chloride inhibition of plant growth is one of the
oldest and most common agricultural problems in the world
(16, 26). Nearly every irrigated region on earth, to some
extent, is affected by high salt a c c u m ulation (26, 29, 90,
9 9, 72, 73). Each year, many ac res of valuable farml and 1 n
the United States are taken out of production or will suffer
severe crop damage b e c ause of high soil salinity or sodicity
( 79 , B2 ) . Sod ium toxicity has bee o me a p r o blem on 50*/. o f
the irrigated land in the western United States, actually
r e stricting crop p r o d u c ti on on 25*/. of this land (20, 72, 73,
75, 85). Moreover, b e cause of excess salinity, on
1
2
millions of hectares of arable land throughout the world, it
will soon become difficult to produce food crops e c o n o m i
cally (01, 82). Many attempts have been made to alleviate
soil and crop losses from high salinity; however, little has
been accomplished. In mast cases, the d e s a 1 i n 1 zat 1 on of
irrigation water or the r e clamation of saline or sodic soils
is impractical and cost p r o h i b i t i v e (EB, 1O 1). The opening
of a g r i c u 1tura 1 1y marginal lands to p r o d u c t i o n is an equally
ex p e n s i v e and resource intensive program which often leads
to u n a c c e p t a b l e losses of valuable native habitat. One e x
ample of this is the d e s t r u c t i o n of much of the Costa Rican
tropical rainforest habitat.
Many researchers believe that tissue culture tech
niques can be used along with conventional breeding methods
for developing salt tolerant crops ( E 0 t 85, 100).
D e m o n s t r a b l e NaCl tolerance has been reported in tissue c u l
tured cell lines of many cultured plant species (IE, El, 83,
2*+, 2b, 3b, bE, 71, 7*+, 85, BO, 100, 106). In addition to
salinity resistance, other stress tolerant cell cultures
have been obtained (1*4, IB, 20, 21, 25, 39, 99, 102). In
order for an agricultural breeding program to be successful,
however, plants must be produced which have commercial or
aesthetic applications. Breeding programs which utilize
cell c u l ture techniques must strive for these goals.
Although r e ge n e r a t i o n from N a C 1-resistant cell lines of
many plant species has been achieved <20, 5A , 68* 72, 73,
75 * 76 * B A , B A , B 5 , 86, 1O O , 103), permanent heritable NaCl
resistance in plants regenerated from tissue cultured cell
lines has not been reported (72,73,75). Nevertheless, r e
searchers are optimistic that with continued efforts, they
will be able to produce cultivars which transmit increased
salt tolerance to their offspring (20, A3, 70, 72, 73, BA,
B 5 , 100). In the following review of the literature, the
effects of salinity stress on plants and some contributions
of tissue and cell culture to recent studies of the
genetics and physio l o g y of plant adaptation to environmental
REVIEW OF THE LITERATURE
□ne of the unifying concepts of environmental stress is
cel lular dehyd ration ( 93 > . Many of the effects of salinity
stress are manifested similarly to the effects caused by
drought stress <97). Pl a n t s suffering from cellular d e h y d r a
tion are wilted, stunted, and in extreme cases, necrotic
(97). Few plants are obligate halophytes, or can tolerate
extremely high salt c o n c e n t r a t i o n s <10). According to
Bernstein (10), even salt resistant plants lining in e n
vironments with a soil conductivity greater than 9 mmho/cm ‘
in the soil solution extract, will be affected by high
salinity.
The major ions cont r i b u t i n g to soil salinity are Cl •
SO.,'' , H C O V , N a ‘ . Ca;‘' , and Mg1" <10, 109). Of all the
ions r e sponsible for salinity, Na' and Cl cause the most
severe crop damage (16, 36, 93, 75). Sodium chloride
toxicity alone affects over one-third of the irrigated land
w or ld - w i d e (16, 69, 73, 73).
High soil salinity results in soil solutions with low
water potentials. Few plants can maintain a negative o s
motic gradient against an external medium with a low water
potential (31, 39, 83). Many plants are able to avoid
9
5
injury or death by e q u i l i b r a t i n g their internal water
p o t e n t i a ls to that of their environmen t . Many salt s e n s i
tive p la n t s are able to m a i n t a i n favorable water activity
for turgor pressure and cell e l o n g a t i o n by taking u p « or
synt h e s i z i n g o s m otically active solutes from the external
medium ( 10, 31 , 3*+ , 37, 97, 99, 95, 96, 97) . Sodium and
p o ta ss iu m are the main c a tions r e sponsible for o s m o r e g u l a
tion, but at high concentrations, they can strongly inhibit
en z ym e and other metabolic activities <32, 33, 3 B , 79, 96,
97). When radical osmotic adjustments are required, plant
metabolic processes are subject to stress.
The effects of salt on plant growth, development, and
p r o d u c ti vi ty have been e x tensively studied (10, 31, 32, 37,
9B, 99, 69, 81, B 2 , 89, 93, 101). Damages due to excess
soil and water salinity still remain a serious concern to
agriculturists. Excess salinity profoundly affects the
biochemistry, anatomy, and morphology of plants. Sufficient
information to cor r e l a t e s a l t — induced alterations to
specific metabolic events, however, is present 1y u n a v a i l
able (01). Accor d i ng to St rogonov < 98) and more recently,
W a l s e 1 <109), tnese c h a nges i n c 1u d e :
a. increased succulence;
b . changes in number and size of stomata;
c. thickening of the cuticle;
6
d . ex tens i ve deve 1opmen t of I y 1a s e s ;
p - ear lier occur re nee of ] i g m f icat ion!
f . inhibition of differentiation;
g. c h a nges in diameter and number of xylem vessels; and
h. stunting at various levels of organization.
In addition to structural alterations, plants affected
by salt stress undergo numerous biochemical changes. A c
cording to Pol j a k o f f-Ma y b er (91 , 92) and Jennings < *9 ) ,
these changes are:
a. increased respiration;
b. decreased germination;
c. reduced CO,-, a s s i m i l a t i o n (due to stomatal closure);
d. decreased p r o tein content;
e. reduced water flow through the plant;
f . 1owered water activity;
g. reduced t r a n s 1o c a t l o n of photosynthate!
h. decreased nutrient availability; and
l. a l t e r a t i o n s in memb r ann per m e a b i 1 i t y .
Damages caused by excess NaCl may be osmotic, toxic, or
nutritional (10). Osmotic stress causes severe internal
plant water deficits. These conditions lead to reduced
water activity, a l t e r a t i o n of m a c r o m o 1e c u 1ar structures,
7
c on c e n t r a t i o n and p r e c i p i t a t i o n of ion;,, and reduced rates
of photosynthesis* cell division and cell expansion (97,
98). Specific ion toxicity occurs when the concentratlon of
nsiTiotical ly active solutes accumulated to effect o s m o r e g u l a
tion become too great <1C). Bernstein <10) describes the
major nutritional effects of salinity stress as those a s
sociated with cation nutrition. With increasing salinity,
c o m p e t i t i v e ion ab sor p tion favors the uptake of Na ’ (10).
In many cases* salt-induced nutritional stresses can be
overcome by additions of C a '' * ( 1O ) .
Plants differ widely in their mechanisms to tolerate
excess salinity (2* 26* 27, 3 1, 39 , 95, 99, 96, 97). Plants
are separated into two catego r i e s based on their abilities
to resist salt stress: h al op h y t e s and g l ycophytes (31, 27).
H a l o p h yt es are plants which increase in dry weight content
in the presence of high conce n t r a t i o n s of electrolytes (at
least 300 m M ) in their environment, while showing no
obligate requirement for high salinity (lO, 31).
Ha l o p h yt es which accumu l a t e salts to effect osmotic a d
justment are called e u ha l o p h y t e s (10, 31). Halophytes which
accumulate organic solutes such as praline, betaine* and
choline for osmotic adjustment are known as qlycahalophvtes
(10, 31). Glyco p h y t e s are plants which cannot survive in
salt sol u t i o n s greater than 0.5*/,. Some, however, do have a
a
limited ca p a b i l i t y for a s m o r e g u l a t i o n (37, 95, 92, 97).
One of the most d e le te ri ou s effects of sodium chloride
stress on crop plants is stunted growth (10). Salt s e n s i
tive plants remain stunted in spite of osmotic adjustment or
ion c a m p a rtmenta 1i z a t i o n . P o 1 jakoff-Mayber (02) and J e n
nings (99) suggest several possible reasons for this: ( 1 )
energy acquired from photo s y n t h e s i s is diverted to active
ion uptake and compar tmenta 1 i z a t io n , syn t h e s i s of organic
salutes, and m a i n t e n a n c e of osmor e g u l a t o r y mechanisms; (2)
the high energy costs of repairing damage to enzymes and
tissues resulting from d e h y d r a t i o n and salt ions; (3)
stomatal closure resulting from temporary loss of turgor
which restricts CO;' uptake; and ( 9 ) dec r e a s e s in anabolic
nitrogen m e ta bo l i s m resulting from all of the above.
According to several authors (17, 31, 3B, 9 9, 50, 82)
NaCl stress alters p r otein synthesis. Interference with
protein synthesis may occur at several levels since: (1)
membrane p e rm ea b i l i t i e s are altered by NaCl, preventing u p
take of organic nitrogen and other n i trogenous compounds
(90, 99, 82); (2) an excess of NaCl lnh ibits at least some
enzyme activity, diss o c i a t e s r ibosomes (11), and leads to
reduct i o n s in p o l y r i b o s o m e complexes (33, 99, 52); (3) NaCl
may cause permanent changes in the genome of plants, a l t e r
ing the synthesis of RNA and the translation of proteins
9
(11, 52, 67, 101); and (A) plant hormones are affected by
salinity which could lead to protein alterations (11* BE).
Tissue and cell c u lture techniques have become useful
to plant breeders for the isolation of p o t entially lmpo r t a n t
p h e n o t y p e s (EO, 70, 100). Another a pp l i c a t i o n of tissue
c ul tu re is in physiological studies for investigating the
tissue and cellular basis of stress tolerance. This a p
proach is not without limitations. According to Tal (100),
using cell culture techniques in studies of this nature has
several unique problems associated with them. One of the
problems is that variants in culture may be expressed only
at the cellular or tissue levels of organization, and not in
whole plants. Likewise, variability may be an epigenetic
change resulting from the selective agent in the absence of
which there is no phenotypic expression. Another problem
assoclat ed with the appli c a t i o n of in v i tro t ec h niqu e s is
the progr e s s i v e decrease in r e g e n e r a b l 1 lty exhibited by
cells and tissues in c u l ture for extended periods of time.
A fourth i nh er ent p r o b l e m associated with virtually all
tissue c u lture research is the absolute necessity for
screening at the whole plant level of crganilation.
The high incidence of variation in plant tissue and
cell c u l t u r e s is a useful trait. Model systems designed for
the investigation of genotypic and phenotypic stability
io
under normal and stressed conditions take advantage of this
p h e n o m e n o n (61, 70). fhe two major sources of heritable
cellular variation are, mutation and epigenetic change
(70). V a riability from s p o ntaneous or induced mutation is
rare and the me c h a n i s m s c o ntrolling epigenesis are still not
fully understood.
The primary source for obtaining phenotypic diversity in
culture, according to Meins (70), is selection. In recent
years, tissue culture scientists have exploited this p r i n
ciple to screen vast numbers of plant cells for desirable
traits <61, 66, 100). Differen t i a t i n g b e t ween physiological
adaptations, epigenetic changes, and directed genomic a l
terations is a fundamental problem inherent in this type of
research ( 19, £?0, 61 , 70, 100) . A method for distinguishing
the e f fects of these sources of variability on phenotype
would be of great practical advantage to plant breeders.
It has been well e s t ablished that the combination of
gene action, epigenetics, and environmental interactions
d e t e r m in es phenotype (IS, 100, 101). How these factors r e
late to salt tolerance still is uncertain. It is likely
that plant responses to salinity stress are regulated at
both the cellular and molecular levels <11, l'?, k3, 100 ,
1 0 1 , 1 0 5 ) .
The c h anges which occur in plants from excess salinity,
11
such as an increased synthesis of o r g a n i c solutes, are often
considered physiological a d a p t a t i o n s that may have survival
advantages under these condit i o n s <B1 ) . These changes have
alterna t i v e l y been d e scribed as pe r t u r b a t i o n s of normal m e
tabolic activi t i e s of living o r g a n i s m s and represent damage
imposed by saline c o n d i t i o n s (BE, 101). Whichever of these
e x p l a n at io ns most accurately a c c o u n t s for the changes in
plants in response to excess salt is not known.
A unique class of polypeptides, o f t e n referred to as
"shock proteins" <9, 51) has a t t r a c t e d a great deal of a t
tention recently for their puta t i v e r o l e in environmental
stress tolerance. These proteins a r e synthesized in many
plants and animals in response to environmental changes. It
is unc e r t a i n when and where the p r o t e i n s are synthesized,
how long these proteins remain in the absence of selective
pressure, and what the nature of their genetic regulation
l s .
Numerous articles report substantial, guantifiable
changes in the p r o te in complement of s o m e plants and animals
in response to various applied physiological stresses (4, b,
11, IP, 15, PE, 51, 53, 107,). Several of these studies
cite p r otein changes directly a t t r i b u t a b l e to osmotic stress
(A, E9 , 30, 91). These supposedly s t r e s s-induced proteins
are synthesized in callus tissue and at the who l e plant
12
level of o r g a n i z a t i o n in response to c e r tain changes in the
environment. Heat, salt, a n a e r o b i o s i s » drought, and cold
are among the stresses eliciting the induction of novel
p rotei ns from numerous plants and animals. It is not known
whether shock proteins actually enhance the ability of an
o rganism to tolerate radical changes in their environment,
however, is not known.
A common theory explaining stress tolerance is not
available. The ability of plants to withstand stress may be
the result of gene action, epigenetic interactions and
physiological adaptation, or spontaneous or directed m u t a
tion. Enhanced ability to withstand stress may be
ephemeral, or transferable, but it is certainly species
spec 1 fic .
One of the most co m m o n methods for the analysis of
proteins in solution is polyac r y l a m i d e gel e l ectrophoresis
(PAGE). Electrophoretic techniques presently are being used
in num erous laboratories for the analysis of the proteins in
cells and tissues of cultured plants. The a p p lication of
these techniques e n ables researchers to determine a great
deal of q u a l i t a t i v e and q u a n t i t a t i v e information concerning
the nature of proteins in solution. One-dimensional sodium
d o d e c y 1 sulfate gel e l e c t r o p h o r es is (SDS PAGE) is the most
widely used technique for p r o tein analysis (^1, 95). The
1 3
type, number, relative abundance, and molecular weights of
p ro t e i n s in a complex m i x t u r e can be identified us 1nq 5DS
PAGE. W he n sodium dodecyl sulfate (an ionic detergent) is
reacted with proteins before electrophoresis, every 1.9
grams of SDS will denature and bind approximately 1 gram of
pr otein (91, 92>. These S DS - p r o t e i n complexes are generally
soluble and ( in an electric field) migrate towards the anode
through an a c r y 1 amide —b 1 s a c r y 1 amide gel matrix. The rate of
m i gr at i o n is generally inversely proportional to the
logarithm of these sds- p r o t e i n c o m p l e x e s ’ molecular weiqhts
(91, 92 ) .
Two factors det e r m i n e the mobility of proteins in an
el e c t r o phoretic system. First, the strength of the electric
field has a direct effect on the speed of p r o t e i n migration.
The second factor is the frictional compon e n t s in the qel
and the shape of the individual proteins. The higher the
viscosity of the gel, the more resistance a protein will e n
counter. The larger and more globular a p r otein is, the
greater the resistance it will encounter <91, 92). The
positions of known molecular weight markers within a gel can
be used to e s ti ma te the molecular weights of unknown
proteins .
Although numerous SDS electrophoretic protocols are
available, the method of Laemmli (60) is the most widely
1 9
used. Laemmli developed a two part gel system which
resolves p r o tein bands better than previous systems which
utilize a one part gel only. The acrylamide c o n centration
of the top or stacking gel is less dense (2-5'/.) than the
acryla m i d e content of the separating or running (l o w e r ) gel
(5-207,J. This differential c o n ce n t r a t i o n gradient favors
increased p r otein band resolution b e c a u s e proteins will
"stack" together at the interface of the two q e 1s
(l s o t a c h o p h o r e s i s ) before they migrate through the running
gel. The c o n c e n t r a t i o n of acrylamide and N ,N ’—m e t h y 1ene
b i s a c r y 1 amide (bis) can be adjusted to facilitate optimal
protein m i g r a t i o n d e pending on the molecular weights of the
proteins being assayed. Some recent studies using SDS PAGE
have linked substantial changes in the banding patterns of
extracted proteins from cells of some species of N i c o t i a na
tabacum with increasing levels of N a C 1 adapta t i o n (29* 30*
91* 107). There has been no evidence linking changes in the
p o l y p e p t i d e complement of cells having N a C 1-reslstance with
a survival a d v antage under high salt conditions, though the
a s s ociation is oft e n made (29, 91).
E f forts towards increasing N a C 1 resistance in crops
should be continued. It has been d e m o n strated with tissue
culture that stable v a riation can be obtained. Cells and
tissues in c u lture inherently exhibit extremely high rates
15
of spontaneous, persistent, and her i t a b l e variation, which
ai e cften transferable in sexual crosses (70) . There is
reason to believe that r e searchers utilizing tissue culture
techniques and conventional breeding methods will be able to
develop superior cultivars.
N 1c o t i a n a tabacum spp. (a dicotyledon) has t r a d i
tionally been used for investigations into the tissue and
cellular mechan i s m s plants have for toleratinq environmental
stress <18, 1 A , 30, 51, 73, 100). Many of the w o r l d ’s most
important horticultural crops, however, are m o n o c o t y 1e d o n s .
Moreover, cereals (also monocotyledons) are among the
w o r l d ’s most important food crops. Creeping bentgrass <A._
pal us t r is Huds.) is a mon o c o t y l e d o n o u s plant. particularly
well suited for use in model systems designed for the study
of i.n v itro stress physiology. Its hormonal, nutritional,
and environmental r e q u i rements for tissue and cel 1 culture
have been established (3, 55, 56, 57, 58). This genus c o n
tains some relatively halatolerant cultivars, but it is
generally considered a glycophytic group of plants with
limited g e netically control led ion e x clusion mechanisms <1,
0, Callus p ro l i f e r a t i o n and plant let regeneration have
been reported from at least four cul t i v a r s of this plant
species (*f6, 57, 58).
A g r o s tis palustris cultivars are highly regarded as
horticultural crops. They are extremely tine turfgrasses
with many des i r a b l e traits and are extensively used as golf*
tennis and bowling greens (0). Creeping bentgrass grows
from vigorous stolons and is one of the most hardy cool
season turfgrasses (0). When mowed closelyi A . p a 1 u s t r i_s
for ms one of the flnest quality sods known < 0 ) . A cultivar
with strong persistent salinity tolerance would be a valu
able commodity.
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IN VITRO SEL E C T I O N FOR S O D I U M CHLORIDE RESISTANCE,
REGENERATION, AND S O L U T I O N CULTURE E V A L U A T I O N S OF
TWO CULTIVARS OF Ag r o s tis p a 1 u s t. ris Huds .
Ab s t r a c t :
Calli from two cul t i v a r s of creepinq he n t g r a s s (Aq r os t 1 s
plustjri^s Huds.) were selected for enhanced ability to
p ro li te ra te on media supplemented with 51A mM N a C l . The t o
tal s o luble p r o t e i n c o n c e n t r a t i on of calli s e 1ec ted from
control media (0 mM NaCl) was greater than soluble protein
c on ce nt ra ti on of calli selected from NaCl s t r e s s - 1 n d u c 1 nq
media. Non- s e l e c t e d calli from N a C 1-stressed and control
media contained less soluble protein than selected calli
exposed to the same treatments. Percent dry weights of
lyophilired callus tissue selected from the control medium
were lower than percent dry weights of 1y o p h i 1 lzed callus
tissue selected from 51A mM N aC l- su p p l e m e n t e d media.
Growth of plants r e g enerated from N a C l - a da pt ed calli in
saline liquid nutrient c u lture <as dry weight and vigor) was
superior to that of plants regenerated from non-adapted
calli. Plants regen e r a t e d from salt s en si t i v e calli died or
were un a b l e to p r oduce m ea su r a b l e dry matter in liquid c u l
ture containing 257mM NaC 1 , whereas pl a n t s regenerated front
N a C l - a d a pt ed calli grew well in 257 mM NaCl* Growth of
33
3^
plants regenerated from salt-adapted calli was inhibited at
NaCl c o n c e n t r a t i o n s greater than 3^0 m M . At 51^ mM NaCl,
plants regenerated from salt resistant calli could no longer
produce measur a b l e dry matter, although they remained alive.
Abb r e v i a t l ons : 2,^-D: 2 , '♦-di ch lorophenoxyacet ic-ac id , B6: N-
(p h e n y 1- m e t h y 1 )- 1- H - p u r i n e - 6 - a m i n e , MS: Mur a s h i g e and SIooq
(5) ,P M S F : p h e n y 1 methy 1s u 1f o n y 1f 1u o r i d e , EDTA (disodium
salt): e t h y 1enediamene t e t r a a c e t i c acid, D T T : d i t h i o t h r e i t o l ,
HEPE5: N - p - h y d r o k y e t h y 1piper a 2 l n e - N '- E - e t h a n e s u 1fonic acid,
TR1S: t r i s t h y d r o x y m e t h y l )a m l n o m e t h a n e , Tween 20: P o 1yoxy-
e t h y l e n e sorbitan monolaurate.
35
INTRODUCTION
Many r e searchers b e l i e v e that tissue c u l ture techniques
can be used along with conventional breeding methods for
d e v e loping salt tolerant crops (2, 13. 15). Several species
of plants have already b ee n regenerated from sodium chloride
resistant cell lines (2. £ , 7 , 9, 11, 10, 12. 13). In vitro
r e g e n er at io n of salt resistant plants from liquid suspension
(9, 19) and callus cul tures (9, 10, 17) has been achieved;
however, permanent NaCl tolerance has not been accomplished
from tissue cultured cell lines. R e s e a r c h e r s are optimistic
that continued e f forts will produce cul t i v a r s transmit
he r i t a b l e salt resist a n c e (2, 11, 13, 15).
Cereals are among the most important food crops in the
world, and like most other monocots, do not tolerate hiqh
c o n c e nt ra ti on s of NaCl in the soil solution. One objective
of this study was to investigate the p os s i b i l i t y of s e l e c t
ing callus lines of the monocots, creeping bentgrass
( Aqrost i_s p a l u s tr i s Hud s. > which can d e m onstrate an enhanced
ability to proli f e r a t e on media supplemented with NaCl and
to s u b s e quently regen e r a t e plantlets from these calli.
Another obj e c t i v e of this research was to evaluate rooted
plants regenerated from NaCl - a d a p t e d calli in liquid
36
nutrient culture for any enhanced ability to tolerate NaCl
stress at the whole plant l e v e l .
M A T E R I A L S AND METHODS
C allus es t a b l i s h m e n t
Callus cultures of 'Penneagle' and ' S e a s i d e ’ creeping
b e n t q r a s s were e s t a b l i s h e d on modified MS medium (5),
sup p l e m e n t e d with 1 mg/1 BA, 5 m g /1 P,6-D, 30 g/1 sucrose,
and 10 g/1 agar. The pH of the media was adjusted to 5.7
with 2 N NaOH prior to a u toclaving (1 k g /c m ; , 121° C for 15
minutes), checked and readjusted after autoclaving if n e c e s
sary. Car y o p s e s were s u r face sterilized by immersing them
in 95*/. ethyl alcohol for 5 minutes followed by immersion in
1.05*/, sodium h yp oc h l o r i t e ( 20'/. commercial bleach solution),
sup p l e m e n t e d with 0.01 7. Tween 20 (surfactant) for 20
minutes. Following s t e r 1 1 i z a t i o n , car y o p s e s were rinsed 3
times in sterile, double distilled water. Two to 5
ca r y o p s e s were e s t ablished in 25 x 150 mm c u lture tubes on
25 ml of medium and incubated in the dark for 30 days. A 1 1
c ultures were cleaned of debris, such as hulls from
car y o p s e s and any eti o l a t e d germinating shoots after 30 days
and transferred to fresh a medium. The remaining callus
material was incubated for an additional 30-days in the
dark.
37
Sel e c t i o n for Salt T o l erance
Stock calli from caryopses, weighing a p proximately 50-75
mg ,were subcultured onto fresh media supplemented with
either 0 or 51^ mM (30 g /1 ) NaCl. Ac tively prol lfpratinq
calli (and portions o+ calli) from both treatments were
selected, rated, and transferred following a sinqle 60 day
dark incubation period. Calli were selected for N a C 1 -
re s i s t an ce based on visual o b s e r v a t i o n s (Figure 1). Those
calli which had not increased in mass beyond their initial
transfer size were given a ( + ) rating. Those calli which
had visibly increased in mass, but less than double their
initial transfer size were given a (++) rating; and those
calli which had doubled their initial transfer size or
greater were g i ven a (+++) rating. Calli which had no
necrotic areas were g i ven a < + > rating; those with 1-25 V.
necrosis a (++) rating; those with 26-50*/. necrosis, a <+++);
and those with greater than 51*/., a (+ + + +) ratinq. Calli
with shoots were given a <+) rating; and those without
shoots a ( — ) rating.. The same rating system was applied to
calli developing roots. All ratings given as (+ or -) were
transformed into p er c e n t a g e s of the original number of c u l
tures in each treatment at the beginning of the transfer
period for subsequent data analysis.
30
E x t r ac ti on and e s t i m a ti o n of soluble proteins
Calli were selected at random from each treatment at
the end of the each transfer period, frozen in liquid
nitrogen, l y o p h i 1 lzed overnight, and stored at -00° C until
needed for p r o tein extraction. One gram of each l y o p h i 1 ized
sample Mas ground with a Thomas motorized tissue homoqeni;er
at full speed in 10 volumes ot chilled a c etone (- ? 0 ° C ) in an
ice bath, suction filtered, and placed in a desiccator
(under vacuum) until all of the acetone had evaporated.
S o luble proteins were ext r a c t e d with a Thomas motorized
tissue homogenizer in an icebath for 30 sec from 100 mg
samples of the acetone powder in 2 ml of lysis buffer c o n
taining 50 mM HEP E S (pH 7.5 at A ° C), 65 mM DTT, 17 mM 2 —
m e r c a p t o e t h a n o 1, 2 mM EDTA, and 1 mM P M S F . The homogenized
samples were transferred to 30 ml Corex c en tr i f u g e tubes and
c en t rif uqed a t 5 0 » GOO x G for 20 min at P ° C. Supernatants
were then car e f u l l y pipetted into screw top microcentr ifuqe
tubes for storage.
One hundred ul a li q u o t s were removed from each of the
samples for p r otein quantification. The remaining soluble
p r o tein extract was frozen in liquid nitroqen and stored at
-00° C far subsequent analyses. Total soluble protein in
each sample was d et er m i n e d by the B i o —Rad protein assay
method. Bovine gamma g l o b ul in was used to generate a
39
standard c a l i b r a t i o n curve for p r otein quantification. A b
sorbance at 595 nm was read usi ng a Bausch and Lomb
Spectranic 21 spectrophotometer.
L y o p h i 1 ized weight d e t e r m in at io n
Each of the stock callus c u l t u r e s generated from caryopses
was divided into ap p r o x i m a t e l y equal segments weighinq b e
tween 75-100 mg and labeled for identiflcation ( i .e. 1 and
1 ’). One segment from each pair was exposed to N a C 1 and
the other segment was used as a control . Since each of the
callus pairs origin a t e d from a common callus culture. they
were referred to as sister c a l l i . For example, cultures 1
and 1 ’ wouId be sister calli; 2 and 2 ’ would be sister
c a 1 1 l . At the be g i n n i n g of each weeL , calli from NaCl
treatments selected for transfer and its co r r e s p o n d l n q c o n
trol were chosen at random, placed in two petri dishes and
mixed thoroughly. Sodium chloride treated calli were placed
in one petri dish and control calli were placed in another.
Two gram samples from each treatment were collected, frozen
in liquid nitroqen, and lyophilized. L y o p h i 1 i zed weiqhts
were compared at the end of three transfer periods.
Plantlet r e g e n e r a t io n and establishment
At the end of three passages, s a l t - adapted and non-
AO
adapted calli (controls) were transferred to r e g e n eration
media. The r e g e n eration medium had the same components as
callus 1 n d u c t 1 on med i u m , ex cep t for the omission of an auxin
source. Calli selected from N a C 1- s u p p 1emented media were
r e generated with NaCl in the growth medium. Controls were
regenerated on reg e n e r a t i o n medium without NaCl. Plantlets
that were regenerated from salt-adapted calli before the end
of the third genera t i o n were set aside and kept on fresh
N a C l -s up pl em en te d media. Embryogenic cultures from all
three g en e r a t i o n s were placed on r e g e n e r a t i o n media without
NaCl for shoot elonga t i o n at the end of the third transfer
period. Plants were acclimated when the majority of shoots
had elongated beyond 20 mm and roots had elongated beyond 10
mm. R e g enerated pla n t l e t s were acclimated off by removinq
culture tube caps and exposing the succulent plant tissue to
the atmosphere. A cc l i m a t i o n took 3 days. Acclimated plants
were estab l i s h e d in 3 inch plastic pots filled with s t e r i l
ized v e r m i c u l i te. Potted plants were placed inside of p l a s
tic ch ambers with ap p r o x i m a t e l y 90*/. relative humidity for
five days, before being moved into a growth chamber. Two
grams of Mi 1o r g a n i t e , a slow release fertilizer, was used as
the source of nutrition. Plantlets were watered with d i s
tilled water every 3 days. This schedule was followed for 1
m o n t h .
U !
L iquid nutrient evaluation
E stab l i s h e d plants were evaluated in hydroponic c u l
ture using H o a g l a n d ’s #2 nutrient solution as modified by
P eterson (13), supplemented with 0 or 51^ mM NaCl. Controls
were acclimated to saline nutrient c u lture in a stepwise
fashion, beginning with 85 NaCl mM the first two weeks, 170
mM NaCl the second and third weeks, 257 mM NaCl the fifth
and sixth weeks. As all control plants were dead at the end
of the sixth week, the liquid nutrient solution was not a d
justed to 51L mM NaCl for the seventh and eighth wee)s for
the controls. Plants regenerated from salt-adapted calli
were acclimated at different rates. The results from
p r e l i m i n a r y experiments indicated that plants regenerated
from salt-adapted calli could tolerate radical chanqes in
the osmotic potential of their medium. The initial
hydroponic medium they were initially exposed to contained
257 mM NaCl. During weeks three and f o u r,plants regenerated
from s a l t - adapted calli were placed in 3^0 mM NaCl, fallowed
by 51A mM NaCl for the fifth through eighth weeks. Two inch
(ID) polyvinyl chloride (P V C ) pipes with holes bored in the
tops were used to support the plants while their roots were
bathed in nutrient solution (Figure 1). Sump pumps were
used to d is tr i b u t e nutrient solutions through the PVC pipes.
The nutrient solutions were aerated
ge
by gravity as the liquids fell back into the reservoirs.
Visual o b s e r v at io ns of the plants were made daily- Growth
was determi ned by dry wel gh t . Each clump of plants was
clipped to a height of 10 cm, and the clippings dried
weiqhed, and recorded. This procedure was repeated for 0
weeks, unless the plants had died. All of the evaluations
were conducted in growth chambers with 16 hours light at 06°
C temperature, and 0 hours of dark at 18° C temperature.
Liqht intensity inside of the growth chambers was 5000
m ic r o E i n s t e l n s /c m ■V s e c . No humidifiers were used. Relative
humidity was a p proximately 0 0 ’/..
RESULTS
E f fects of Salt
Sodium c hl o r i d e had a highly significant effect on the
percent of callus cultures transferred (Table 1). At the
end of the first generation, 95.3'/. of the non — stressed
’P e n n e a q l e ’ c ul t u r e s and 90.3% of the ' S e a s i d e ’ cultures
were selected for transfer. Of the cultures transferred,
92.77, of the nan-stressed 1 Penneagle ’ cultures and 967. of
the ’S e a s i d e ’ cultures were selected for transfer a second
time. At the end of the second transfer period, 90.77, of
the ' P e n n e a g l e ’ cultures and 86.77. of the ' S e a s i d e ’ cultures
43
were selected and transferred. After the first g e n e r a t i o n
on NaC 1 -supp 1 emented medium, 00.7V. of the 'Penneagle' calli
could be transferred and 66V, of the 'Seaside' cultures were
selected for t r a n s f e r . At the end of the second transfer
period, only 7 V. of the ' P e n n e a g l e ’ calli and A . 1V. of the
' S e a s i d e ’ cultures were suitable for transfer to fresh
m e d i um for a third generation. A total of B1V. of the callus
cultures an control media were transferred during this e x
periment over three g e n e r a t i on s and used in the regeneration
studies, while only 0.02V. of the callus c u l t u r e s exposed to
514 mM NaCl remained after the third selection/transfer and
were s uitable for the r e g e n e r a ti on studies.
There was a highly significant difference between the
number of cultures producing shoots and roots on control
m edium and the number of cultures producing shoots and roots
on a N a C 1- s u p p 1emented m e d iu m (Table 2). Sodium chloride
depressed the p r o d uc t i o n of itinerant shoots (in both
cultlvars) on callus m a i n t e n a n c e medium (Table 2). Neither
cultivar produced shoots on 514 mM N a C 1- s u p p 1emented
medium, but both cu l t i v a r s produced roots on the 514 mM
NaCl media. There was a highly significant inhibition of
root pr o d u c t i o n by calli of both cultivars on the NaCl-
med ium (Table 2).
Salt also had a highly significant effect on the
AA
visible increase in mass (size) that both cu l t i v a r s gained.
Both c u ltivars produced s i gnificantly less mass increase on
media supp l e m e n t e d with SlAmM NaCl than an control media
over three g e n e r a t i o n s (Table 3).
Growth media s u p p l emented with 5 1 A mM NaCl had a highly
significant effect on n e c r o s i s over 3 generations. There
was c o n s i d e r a b le variability among cultivars and the p e r
centage of necrosis. ' P e n n e a g l e 7 had the greatest number of
cultures with zero necrosis (Table A) over 3 transfer
periods and ' S e a s i d e ’ had the greatest number of cultures
with 1-25V, necrosis (Table A). Alternatively* ' P e n n e a g l e 7
p roduc ed the greatest number of c u l t u r e s with 86-50*'.
necrosis (Table A). At the level of 51 percent or greater
necrosis (Table A) there was no significant difference b e
tween cultivar response.
S odium chloride strongly reduced the number of callus
cultures remaining from both cultivars at the end of three
transfer periods. Less than IV. of the initial cultures e x
posed to 51A mM NaCl were selected for r e g e n eration
Sodium chloride had a highly significant effect on the
number of calli from both cul t i v a r s r e g e n erating shoots over
three g e ne ra ti on s (Table 5). There was a significant inter
action b e t ween g e n e ra t i o n time and the number of cultures
pro d u c i n g shoots. More calli from both cul t i v a r s produced
A5
shoots during the first transfer period on r e g e neration
media than during the third generation (Table 5). There was
also a significant d i f f e r e n c e among cultivar and treatment
n N a C 1 or no NaCl ) . Sodlum chl o r i d e - a d a p t e d 'P e n n e a q 1 e ’ c u l
tivars produced far more cultures with shoots on 51A mM
N a C 1—s u p p 1emented r eg e n e r a t i o n media than 'Seaside". while
nonadapted ' S e a s i d e ’ calli produced a greater number of
em b ryoqenic cultures on control media.
So d i u m chloride also had a significant effect on the
number of cultures pro d u c i n g roots. Roots were produced by
both cul t i v a r s in a s i g n i f l c a n t 1y greater number of cultures
on N aC l - s t r e s s inducing media than on control media (Table
5). There was also a highly significant cultivar by salt or
nonsalt interaction (C X S), Seaside' calli produced a s i q -
nifica n t l y greater number of cultures with roots than
'Penneagle* however, 'Penneagle' produced more cultures with
roots on control media (Table 5).
A total of 1 <b2 NaC l - a d a p t e d ’P e n n e a g l e ’ cultures u l
timately produced rooted plants on 51A mM supplemented
media. Only A3 N a C l - a da pt ed ' S e a s i d e ’ calli produced rooted
plants on salt stressed media.
The mean l y a p h i 1i zed weights of the control treatments
were lower than NaCl treatments (Table 6). The protein c o n
c e n t r a ti on in control treatm e n t s was only slightly higher
A 6
per 100 mg of lyophilized tissue compared to protein c o n
c e n t r a t i o n s of lyophilized NaCl treatments. Samples from
selected N a C 1-stressed and non-stressed calli of both cul-
tivars had protein c o n c e n tr at io ns that were significantly
higher than samples from nonselected salt-stressed and non-
s t r essed calli.
Normally regenerated plants of 'Penneaqle' and ' S e a s i d e 1
produced dr y weight more slowly than plants from normally
germinated caryopses or plants regenerated from salt-adapted
calli (Figure E ) . The normally r egener a t ed plants obtained
the same level of g r o w t h ( however, by the end of B weeks.
Plants either from normally germinated caryopses or
regenerated from n o n -adapted calli adjusted enough at 85 mM
N a C 1 to show an appre c l a b l e growth in terms of dry matter
(Figures 3 and A) . At 173 m M , plants from plants from both
of these groups had either died or were showing absolutely
no growth (Figures 3 and A). Plants r e g enerated from both
cultivars of NaCl - a d a p t e d bentgrass calli grew and added dry
matter at E57 mM NaCl (Figure 5), but were strongly in
hibited at NaCl c on ce nt r a t i o n s higher than this. At 51A mM
NaCl they were still alive but unable to p r oduce measurable
dry mat ter .
A7
DISCUSS!ON
The results of this study indicate that it is possibile
to isolate N a C 1 - to 1erant callus cultures of two cultivars of
Aqrostj^ P#J_y?tr_is with enhanced ability to tolerate hiqh
salinity conditions. Sodium chloride at a concentration of
5 1A mM closely approximates the salinity of seawater. This
is an extremely high c o n ce nt ra ti on of saiti and one which
would unlikely be encountered. Figure 6 illustrates calli
of ' P e n n e a g l e ’ (top) and ' S e a s i d e ’ (bottom) which have been
exposed to 5 1^ mM NaCl. There are c e rtain cells or qroups
of cells which appear very healthy (translucent and friable)
and actively p r o liferating at this concentration. One might
expect that plants regenerated from these NaCl-adapted calli
could have some selective advantage to withstand higher c o n
c en t r a t i o n s of NaCl in the soil solution than plants
regenerated from non-adapted calli, or from plants normally
qermin a t e d from caryopses. To some degree, the results of
this study indicate this. Plants r e generated from salt-
adapted calli maintained superior growth compared with n o n
adapted plants at NaCl c on ce nt r a t l o n s up to B57 m M . They
remained alive but did not grow at c o n e e n t r a t ions up to 51k
mM NaCl (Figure 7).
The selection of calli or the r e g e n e r a t i o n of plants
from N a C 1-resistant calli at NaCl concentrations as high as
<40
51 <4 mM has not been reported. Previously, Kochba et al . (5)
obtained C itrus s i nensi s callus resistant up to 170 mM;
Nabors (10, 11) obtained NaCl-tolerant N i c o t i a na tabacum
s p p . at m a x imum c o n c e n t r a t i o n s of 150 mM. Croughan (3),
S a l q a d o - G a r c i g l i a et al. (17), and Tyagi O l ) all selected
for N a C 1-resistance in calli of alfalfa, sweet potato, and
Jimson at 170 m M . It was possible to regene r a t e plants from
s alt-a dapted calli at c o n c e n t r a t i o n s of 51 <4 mM NaCl. Nabors
(11) was not able to r e g e n e r a t e plants from N a C 1 -reslstant
calli unless they were placed on a non-stressed medium
Tyagi et al. (21) r egener a ted NaCl - a d a p t e d D a tur a i nno i a
calli at 170 m M . Mathur et al. (6) obtained regeneration of
K i c k_x i a ramo s i s s i ma at 120 mM NaCl c o n c e n t r a t i o n s from in-
ternodal sections, not from NaCl-adapted calli.
□ne of the more promising aspects of this work is that
calli be c a m e embr yogenic whlie exposed to the s e 1ec 1 1 ve
agent for which they were screened. Acc o r d i n g to Chandler
and Thorpe (2), this is an extremely des i r a b l e trait in
breeding programs for salinity resistance. It is likely,
from their appearance, that plantlets r e g enerated from salt
adapted calli on 51 <4 mM NaC 1 -supp 1 emented r e g e n eration media
were under salt stress conditions. They exhibited the same
symptoms as whole plants grown under salt under salt stress
c ondit i o n s in the field (figure B>. The regenerated
99
pla nt le ts were dark green or dark greenish*blue, succulent,
and stunted (1). Plants regenerated from selected calli
did not show any superior growth in control liquid culture
(Figure 2 > . However, they were superior to plants qrown
from normally germin a t e d car y o p s e s (Figure 3), and plants
re g enerated from non-adapted calli (Figure 9) in saline
hydrop o n i c medium. They appeared have an enhanced ability
for o s m or eg ul at io n under N aC l - s t r e s s conditions.
Pl a n t s regenerated from salt adapted calli of both c u l
tivars, performed similarly in liquid culture, alt. houqh,
' S e a s i d e ’ is consid e r e d to be the more halotolerant of the
two cultivars. It is p os s i b l e that superior 'Penneaqle'
q e no ty pe s were selected and regenerated as a result of the
s c r e e m n q procedure.
It was not possible to grow plants f r o m normally g e r
minated caryopses, nor normally regenerated plants in 519 mfi
NaCl liquid culture. However, plants regenerated from sodium
chloride selected calli were capable of w i t h standing this
shock and recovered. ' P e n n e a g l e 1 calli transferred over 3
generations, therefore may have superior genetic salt r e s i s
tance mechanisms. This sel e c t i o n procedure is a method for
singling out these individuals.
Data from the mean weights from 2.0 g samples of
lyophilized tissue indicates that Na C l - s t r e s s e d tissue is
50
higher than tissue of control calli in dry matter. The
lyophilized weights from selected and non-selected tissue
was not a p p reciably different from their corresponding
selected sister calli. The p r o t e i n c o n c e n t r a t i o n from n o n
selected calii from both treatments were significantly less
than from select ed calli. An e s t i m a t i o n of total soluble
proteins* therefore) may facilitate rapid screening for
callus tissue with enhanced ability to tolerate osmotic
Whether any enhanced ability to tolerate high NaCl is
lost in the absence of the selected pressure or is t r a ns
ferred to progeny is not presently known. Further studies
will have to be performed to det e r m i n e this.
LITERATURE CITED
1. Catarino, F.M. and A.J. Trevawas. 1970* Metabolic
changes in nucleic acids associated with the development
of succulence. Phytochemistry. 9: 1807-1B09.
2. Chand ler , 5. F . and T. Thorpe. 1986 . Var i a t i o n from plant
tissue cultures: B i o t e chnalog ical a pp li ca ti on to improv
ing salinity tolerance. Biotech. Adv. 9: 117-135.
3. Crnuqham, T.P.i 5.J. Stavarek, and D.W. Rains. 19 6 Q .
S e le ct io n of NaCl tolerant line of cultured alfalfa
cells. Crop S c l . IB: 959-963.
9. Epstein* E. 1972. Mineral nutrition of plants: p r i n
ciples and perspectives. John Wiley and S o n s » N. Y.
5. Kochba, J.> G. Ben-Hayim, P. Spiegal-Roy, S. Saad , and
H. Neumann. 1982. Sel e c t i o n of stable salt-tolerant c a l
lus lines and embryos in Clt r u s s lnens i s and C. auran-
tium. Z. Pflanrenphysiol . 106: 1 1 1 - 1 1B .
6. Mathur, A.K., P . 5. Ganapathy, and B .M . J o h r l , 19 B 0 .
Isolation of sodium chlori d e - t o l e r a n t pla n t l e t s of Kick-
x l a ramosjssi^na under in vitro conditions. Z. P f l a n z e n -
physiol . 99 i 207-299.
7. Murashiqe, T. and F. S k o o g . 1962. A revised medium for
rapid qrowth and b io as s a y s with tobacco tissue
cultures. Physiol. Plant. 15: 973-997.
51
8. Nabors, M.W. 1983. Increasing the salt and drought
tolerance of crop plants. p. 165-189. In: D. Randall
< e d C u r r e n t topics of plant bioc hemistry and p h y s i o l
ogy, Vo 1 -P, Univ. Missouri Press, St. Louis, MO.
9. Nabors, M.W. and T.A. Dykes. 1909. Tissue culture of
cereal cultivars with increased salt, drought, and acid
tolerance. p. 181-139. In: B i o technology in internat
ional agricultural research. proce e d i n g s of the inter-
centera seminar on international agricultural research
(LARCS ) and biotechnology. International Research In
stitute Manila, Philippines, pp. 121-139.
10. Nabors, M.W., A. Daniels, L. Nadolny, and C. Brown.
1975. Sodium chloride tolerant lines of tobacco cells.
Plant Sci. Let. 9: 155-159.11. Nabors, M . W . , S.E. Gibbs,
C.5. Bernstein, and M.E. M e i s . 1980. N a C 1 - t o 1erant
tobacco plants from cultured cells. Z . Pflanzenphysiol.
97: 13-17.
12. Nabors, M.W., C.S. Kroskey, and D.M. McHugh. 19B2. Green
spots are predic t o r s of high callus growth rates and
shoot formation in normal and salt stressed tissue c u l
tures of oat (A v e na sativa L .t . Z. P f l a n z e n p h y s i o l . 105:
391-399.
13 .
19 .
15 .
16 .
17.
1 B .
19 .
53
Norlyn, J.D. 1900. Breeding salt tolerant plants.
Bi o s a l i n e Res. S3: S93-309.
Rains, D.W., T.P Croughan, and S . J . Stavarek , 1979.
Sel e c t i o n of salt tolerant plants using tissue culture,
p. 257-S79. In: The genetic engineering of o s m o r e g
u l ation impact on plant p r o d u ctivity for food, c h e m i
cals, and energy. P l e nu m Press, N. Y.
Rains, D.W. 1902, D e v e loping salt tolerance. Calif. Aqr .
31-33.
Rang a n , T.S. and I .K . Vasil. 1983. Sodium chloride
to lerant emb r y o g e m c cell li nes of j V H 1
amer ic an u m (L. ) K. Schum. Annals Bat. 52: 59-69.
S a 1g a d o - G a r c l g 1 i a , R., F. L o p e z - G u t ie r r e z , and N.Ochoa-
Alejo. 19 B 5 . N a C 1 -resistant variant cells isolated from
sweet potato cell suspensions. Plant Cell Tissue Organ
Culture. 5: 3-12.
Tal, M. 1903. S e l e c t i on for stress tolerance. p. 961-
988. In: D.A. Evans, W.R. Sharp, P.V. Ammirato, Y.
Yamada, ( e d s . ) , Handbook of plant cell culture
Vol. 1. M a c m i l l a n P ub li s h i n g Co., N. Y.
Tal, M. 1905. G e n e t i c s of salt tolerance in higher
plants: Theoretical and practical considerations. Plant
and S o i 1. 89: 199-226.
5 9
20. T e m p l e t o n - S o m e r s , K.M., W.R.S. Sharp, and R.M. P f s t e r .
1901. Se l e c t i o n of c o l d - resistant cell lines of carrots.
Z. Pflanzenphysiol. 103: 139-198.
21. Tyagi, A . k'. , A. Rashid, and S.C. Maheshwar i . 19B1.
Sodium chloride resistant cell line from haploid Datura
i n no k a Mill. A resistant trait carried from cell to
plantlet and vice versa. Protoplasma. 105: 327-332.
Taale :. Effects of 5l«t Nall and generation upon transferable call s of calustr:s P e n - p e ; ! e i ' P t ’ .' a n d S E a o i a p ‘ E S 1 .
Ferrentaae if call: tr ir,sferred froa earh qeneratur
Ni ia:t Bolt
tiener at; o -
:uliua 1OS ; , ; p
S3.3-
PB.’T'B.'l re.
“ 0 . 7 . 3 . B
B'.7 3.3.'
o c. 0 7.5 .
M'. 11.r
*1 . . .J,£' it .3.5.1
♦ dear ar.d standard er-cr ■' ftr percentage cultures of k palus.tr :s ’Perioaole' anc 'Be-side'selected and transferred ( o r 3 oeneraticns. Per: pr>t sqes represent data free 3 rep 1:: a 11 :.n-;.
Table 3. Ef fee 15 of IN a!' S t l i or the production o' sheets an: roots '"rest ca.l: n.ito-es :.f ft. p a 1 ur t r 1 s fenne.-tble ■ arid Geaside ■ 'as' ■ on callusi id-..: 11 on a p d i u a Our: rc a r k i n c u D i m n .
Perze'.taSf c u i t i ' e s p r r ea c jn q sheets anc r c c t s £••• c a l l u s mauc t i : r. i s c . e . i
Nv_5dj_! S a i t
_ _ _ _ _ C - e r e r a t if 1 _ _ _ _ _ _ _ _ _ _ _ _ G er e a 11 c r _ _ _
L'.iti.a 1 6 3 1 3 3
•ft ■
Snot- ”s 1.3 116c: ts 33.','1 ; ■ 39.0 L;
9 ,j3t . O . 11/
'j11.3 ■.
911 . 6 3t . r !
1 -ft' 1 1 3h.l :H 1
* Kesr ard s t a i d i ' d e r ' i r i for pe r c e n t c u l t o r e s of p a i u s t - 1 s ' Fen ' ea : ; - : - ' a r : ’ S e a s i d e ‘m: th s t o a t s and r o o t s c a l c u l a t e d at t he bee t ' t i n : of each t r a n s i t
p c r : rd f t ' 3 c e n e - a t i t -s. Fe c e n t a l s r e p r e s e - t da t a ' r o t 3 r e p l i c a t i o n s .
57
Table 3. The effects of 514 mM NaCl on the percentage of cultures of A_;_ pa lustr is Penneagle ('PE') and Seaside ('5S’) which had visibly increased in size.
No Sa1t 5a 11
C v ./Gen.
( + )Callus Growth( ++ ) ( + + + >
Callus Growth ( + ) ( ++ ) (+ + + >
‘PE '
1
2
3
4.0(2) 13.5(2) SI.7(2)
1.8(2) 11.0(2) 87.2(3)
4,2(2) 17.0(4) 7B.8(3)
45.3(15) 41.3(h) 3.3(1)
54.7(4) 42.7(4) 2.7(1)
36.0(7) 62.3(6) 4.B(3)
'SS’
1
2
3
0 6.3(1) 93.7/1 ) 40.7(7)
0 5.7(2) 93.2(2) 44.7(5)
0.07 12.3(1) 87.7(1) 34.0(7)
53.3(7) 3.3(1)
56.0(3) 1.0(1)
62.3(6) 3.7(1)
* Mean and standard error ( ) for percent cultures of A . pa lust rls‘P E ’ and 'S S ’ which had not visibly increased in size beyond their initial transfer size (+>; those cultures which had visibly increased beyond their initial transfer sizei but not doubling (++>; and those cultures which doubled in beyond their initial transfer size (+++>. Percentages represent data from 3 generations and 3 replications/ generation.
5B
Table 9. The effects of 519 mM MaCJ on the percentage of A . palustr1s Penneagle <'P E ’> and Seaside ('SS’) callus cultures with varing degrees of necros 1s .
No Salt Salt
Cv , /Gen.
0*/. Nec r os i s 1-25 26-50 >51
’PE ’
1 88.7(E) 6.3(2) 11.0(1) 0.1
B 95.0(1) 5.0(1) 0.1
3 99.0(3) 5.3(3) 0.7
0
O
’/. Necrosis0 1-25 Bb-50 >51
7.3(E) 19.3(E) 69.3(9) 9.0(5)
11.0(1) 31.3(E) 50.7(1) 7.0(E)
8.7(3) 3E.6(9) 9? . 3(11) 13.0(5)
'SS’
1 96.0(S) 3.0(E) 1.0(1)
E 88.0(E) 10.7(3) 1.9(1)
3 83.3(3) 16.0(3) 0.7(1)
0 9.7(2) 69.7(6) E0.7(0) 3.3(1)
0 5.7(1) 63.7(6) 27.3(6) 6.0(E)
0 9.7(E) 35.3(9) E9.0(3) 30.3(E)
* Mean and standard error for percent cultures of pa 1ustris ’P E ’and ' SS ’ with 0'/., 1-E5’/., 26-50’/.* and >51*/. necrosis over 3 genrations. Percentages represent data from 3 replications.
T a t 1 e 5 , ' t i e e v f t o f 51** ftt* ha C i o n t h e d r o c i . : t i o n o f s h . t t s a n d r o d s f r o n r a l . u s c : : l t u r e s of t n l i : £ t ' i 5 ' V m e a d l p • ' P E ' a n d S e a s i d e ' . ‘ S o t o r r e d e n t r a t i o n * e d i j « .
P e rte-:tai;e of c o ! t u res p r o d u c i n g snoots and re n t s or r e c e r e ra t i o n e e d u a
Ho S a i t S a i l
6 enp* at ion bene- a t : or
l o_: t; v a ’ !_______ 3___ 3 ________ 1________ c ____ 3 _
' f£'
a l ' oc t s s » . " 3- a l . u 1! ' j 5 - j ■ -+ ■ Hn. t i 1- Si 3 e . " ' t : S ? . 3 1 r ■
k o : t s 7 1 . y i r i <* 3 . v 5 3 “ . 7 i i ■ *< 0 . ■ 4 1 4 9 . 1 ' ! ■' 4 4 . ' J 1 1 .
sf.oots :s '■-■,*''0 ‘7“,'7C;1 IS.jj li.t 5 4.!1.**.1Pools Ea.a hO.' • Se.j'ii HE.3’3 "E.vS)
t Hear and standard error it for percent cultures :f palustr 1 s ' F E ’ and 'SS' fro* ttierMtip.a:ec on regeneration aedie wricf; p'odurec snorts and roots c*f 1- oer er a t;o * s. fei:ertfdes represent dat; f:c 3 rephceticrs.
0‘.
Table t . The effects cf 5S •t' Nail or 1 vophiii;ed neiqnt and p-ctem, concert'at i:r. c* selected ;nc no os elected callus cultures of Fenneeq-e i'cE'.' ar.p Seaside 1'55 ■.
Sel ec ted heree!ec ted
P r Ci t e i n
1 [-,1 Trea*»e'' t s.■■ op" - Wt. CO ":C .
uc .1Lytc-,. Wt. Icn: ,
u: ■ u:
’ff
0. le? 3.’
■j.252 l.e
Cc.nt: [■ i
5N»* ' J . CS4 J . Z
1 . 2
l.C
* Mean anc standard error i 1 cf ivt-ptiil i:ea we:oM and protein ccrtent_at it n selected and nonselected h. paiusris 'sister calli" for 3 generations. oscair.ec froa ;.0 arari satples fresf weight tissue.Soluble protens eitracted fro* ;0u aq cf lyophi ii:ed tissue.Standard error for Lvoph: 1:red weig tt p'ctem corccertration r 0 for ail testej.
61
Fig. 1. Visual rating chart for determining percent necrosis of ft.pa 1ustris 'Penneagle’ or ’Seaside’ calli cultured at 0 mM and 51^ mM NaCl. From left to right: 0’/. ( + ), 1-35*/. (++>,36-50*/. (+ + +>, and greater than 51'/. (+ + ++).
62
63
Fig. 2. Dry weight of A_;_ palustr i & ’Penneagle’ and 'Seaside' plants germinated from caryopses ( Q ), plants regenerated from calli cultured on 0 mM NaCl supplemented medium ( ) « and plants regenerated from calli selected from 5ig mM NaCl supplemented medium * X grown hydroponica 1 1 y in 0 mM Haagland’s #2 nutrient medium. Bars represent standard error.
Dfi
Y W
EIG
HT
CAIN
(g
) S
RY WEICHT (g)
64
' PENNEAGLE' O m M
3 -
20 64 1 0W E E K S t N C U L T U R E
O N C C O m M 4 N R F O m M * S E L E C T IO N S O m M
' S E A S I D E ' O m M€
5
4
3
2
1
02 100 6 84
O N C C O m MW E E K S IN C U L T U R E
4 N R P O m M * S E L E C T I O N S O m M
4.5
Fig. 3. Response of A_. paiustr is. 'Penneagle' and 'Seaside'plants germinated from caryopses cultured hydroponica11y in Hoagland’s #3 nutrient medium with 0 mM NaCl < <^>) or with 357 mM NaCl ( Q ). Salinization procedeed i n a stepwise gradient. At week 3 plants were exposed to 05 mM NaCl; at week A plants were exposed to 173 mM; at week 6 plants were exposed to 257 mM NaCl; and at week 0* if any plants were alive, they were exposed to 51 A mM NaC1.
CRY
W
EIG
HT
CAIN
(g
) D
Pr
WE!
a}!T
C
AJf
* (9
)
66
NORMALLY GERMINATED CARYOPSES' P E N N E A G L E '
S
4
3
Z
1
00 2 4 e 6 t o
W E E K S I N C U L T U R E o N C C O m M * N C C ft* m M
NORMALLY GERMI NAT ED CARYOPSES
\7 3
2 . S -
0.6
e 1 o60 z 4
W E E K S I N C U L T U R E D N C C O m M ♦ N C C 5 t A m M
67
Fig. k . Response of A^ pa 1 us t r i s, 'Penneagle' and 'Seaside' regenerated from calli cultured on 0 mM NaCl supplemented medium, grown hydroponica 11y in Hoagland's #2 nutrient medium with 0 mM NaCl ( Q ) or at 51 * mM NaCl ( > - Salinization procedeed in a stepwise gradient.At week 2 plants were exposed to 85 mM NaCl; at week A plants were exposed to 173 mM; at week 6 plants were exposed to 257 mM NaCl; and at week 8, if any plants were alive, they were exposed to 5lA mM NaCl.
DRY
If
EIG
HT
CASH
tg
) D
RY
WE
1C SI
T CA
SH
(g)
68
NORMALLY R E G E N E R A T E D PLANTS' P E N N E A C L E '
€
S
3
2
T
00 2 4 £ 8 1Q
W E E K S C U L T U R E D S R P O m M * M R P S f 4 m M
NORMALLY R E G E N E R A T E D PLANTS€
5
4
3
f
O0 2 4 6 a to
W E E K S i r t C U L T U R E D b l R F O m M ♦ b f R P 5 M mi/
69
Fig. 5. Response of A_ pa 1ustr is. 'Penneagle' and 'Seaside' plants regenerated from calli selected from 51A mM supplemented medium, grown hydroponica 11y in Hoagland's #2 nutrient medium with 0 mM NaCl ( Q ) or at 51^ mM NaCl ( . Salinization proceeded in a stepwisegradient. At week 8 plants were exposed to 85 mM NaC1; at week A plants were exposed to 173 mM; at week 6 plants were exposed to 857 mM NaCl; and at week 8, if any plants were alive, the> were exposed to 51^ mM NaCl.
DRY
W
EIG
HT
CAIN
(g
) D
RY
WEI
GH
T CA
IH
(g)
70
S E L E C T I O N S O m M V S . 5 1 3 m MPENNEAGLE'
6
85 257
S
.j y
J
2
1
02Q 4 G a t o
W E E K S I N C U L T U R E o S E L E C T I O N S O m M ♦ S E L E C T I O N S 5/4 mJ/
SEL ECT IONS O mM V S . 5 1 3 m M€
2 5 7
S
3
Z
1
00 Z 4 6 6 t o
W E E K S I N C U L T U R E D S E L E C T I O N S O m M * S E L E C T I O N S S t A m M
71
Fig. i. Callus cultures from ft. palustris 'Penneagle'(top) and 'Seaside’ (bottom) cultured an a medium supplemented with 51^ mM NaCl. Lighter areas are portions of cultures where cells are actively proliferating. Such areas were selected and transferred.
73
Fig. 7. Response of pa 1ustris. 'Penneagle' (top left), and’Seaside’ (top right) plants regenerated from NaCl- adapted talli cultured hydroponica 11y in Hoagland’s #3 nutrient medium at 0 mM NaCl. On the bottom are A . palustris plants regenerated from NaCl-adapted calli in 057 mM NaC1.
u
75
Fig- 9. ftqrostis palustr 1 s. 'Seaside’ plantlets regenerated on medium supplemented with 51^ mM NaCl. Note succulence, dark color, and stunted morphology of plants as a typical symptom of salt stress.
It
/i*
SY N T H E SI S OF SALT SHOCK PROTEINS IN CALLUS CULTURES OF
flg r o s t i s p a 1 u s t r is H u d s . IN RESPONSE
TO NACL STRESS
Ab s t r ac t :
Callus cultures of Aq r o s tis p a l u s tr is Huds. grown on a
me d i u m sup p l e m e n t e d with 51L mM NaCl produced several unique
pr otei n bands on one dimensional SDS p o l y a c r y l a m i d e qel
slabs compared to calli grown on medium without NaCl. A p
parent increases in the density of some already pxistinq
pr o tein bands and reduct i o n s in the density of others were
also observed. Induced synthesis of novel proteins was o b
served after the second week on N a C 1- s u p p 1emented media.
After sub c u l t u r i n q N a C 1-adapted calli onto media without
NaCl for one transfer period <30 days), the production of
stress induced proteins ceased, or could not be detected on
one dimensional gel slabs.
77
7B
A b b r e v i a t i o n s :SDS PAGE: sodium dodecyl sulfate p o l y a c r y l
amide gel electrophoresis, MB: Mur a s h i g e and Skoog (13),
PMRF : p h e n y 1 m e t h y 1s u 1f o n y 1 fluoride, EDTA: ethylene-
d l a m l n e t e t r a a c e t ic acid d i s o d i u m salt, DTT: d i th lothrei tol i
HEPEB: N - 2 - h y d r a x y e t h y 1piper a 2 i n e - N ’- 2 - e t h a n e s u 1fonic acid,
TRI5: t r i s (h y d r a x y m e t h y 1)a m l n o m e t h a n e , Tween 20: polyoxy-
ethyle le sorbitan monolaurate.
7 9
INTRODUCTION
A stress-inducedi unique class of p r o t e i n s ( often
referred to as ‘’shock proteins"* are presently being r e
searched for a putative role in the tolerance of plants to
environmental stresses <1* 9, 9, 10). Although no strong
co i'relation b e t ween the production of stress-induced
proteins and any increased survival advantage under c o n d i
tions of environmental extremes has been established yet,
the asso c i a t i o n is often made (10).
Much of the current research with "shock proteins" is
in three areas. R e s e a r c h e r s in these areas are seeking to
det e r m i n e the location in the cell and the time when stress
p roteins are synthesized! how long s ho ck - i n d u c e d proteins
remain in the absence of a selective pressure (stress); and
c h a r a c t e r i z i n g the nature of the genetic regulation of the
proteins.
Significant a l t erations in the p r o t e i n c om p o s i t i o n of
many plants and animals in response to changes in their e n
vironments have been reported (9, 5, t, 7, 9* 10, 11, It,
IB). Sever al studies have reported c hanges in p o 1ypep tides
that are directly a t t r ibutable to osmotic stress (7, 8, IB).
Shock or stress prot eins have been observed at the whale
plant level of o r g a n i z a t i o n (11) and in cultured tissues and
80
cells (7, 18).
Nico t i ana to b a c u m , a dicotyledon, has traditionally been
used in model systems for i n vitro stress physioloav r e
sear c h ( 1 A , 15, 19). E r i c k s o n and Alfinito ( 7 ) , and Sinqh
et. al (18) used tobacco suspen s i o n cultures to investigate
p rot e i n s associated with NaCl stress. Many of the w o r l d ’s
most valuable horticultural and agronomic crops, however,
are monocotyledons, and little work has been done to
e l u c i d a t e some of the p r o t e i n changes occ u r r i n g in monocots
as a result of salt stress.
The purpose of this study was to utilize a
m o n o c o t y l e d o n o us flqrostls palustris, to investigate the
synthesis of salt shock proteins contained in the total
soluble protein fraction of NaCl - a d a p t e d and nan-adapted
calli. Two member s of A . p a l u s tr is, 'P e n n e a q 1e ’ , a s a 1 t -
sensitive cultivar and ' S e a s i d e ’, a relatively salt-
tolerant cultivar were compared.
Ma t e r i a l s and Methods
Establi s h m e n t of be n t g r a s s callus
Callus cultures of ' P e n n e a g l e ’ and ' S e a s i d e ’ creeping
bentgrass (Aqro s t i s p alu s t ris H u d s . ) wer e established on
modified MS medium (13), supplemented with 1 m g /1 BA, 5 m g /1
2,A-D, 30 g/1 sucrose, and 10 g /1 agar. Prior to autoclav-
a 1
i nq (1 kq/cm1', 121° C for 15 min) , the pH was adjusted to
5.7 with 2N N a O H . After autoclavinq pH was checked and
readju s t e d with NaOH if necessary. Car y o p s e s were surface
st e r i l i z e d by immersion in 95 Vi ethyl alcohol for 5 minutes
followed by immersion in 1.05*/. sodium h y p o c h l o r i t e ( 20'/. c om-
mercial bleach), s u p p l emented with 0 . 0 1 V. Tween 20
(surfactant) for 20 min. Following sterilization, caryopsps
wer e rinsed 3 times in sterile, d i stilled w a t e r . T wo to 5
c a r y o p s e s were e s t a b l i s h e d in 25 * 150 mm culture tubes and
incubated in the dark at 20° C for 30 days. After 30 days,
cultures were transferred to fresh medium, then incubated in
the dark for an additional 30-days.
Induction of sodium c h l o r i d e stress proteins
At the end of the second 30-day dark incubation period,
callus cultures gen e r a t e d from caryopses were divided into
a p p r o u m d t e l y equal segments weighing b e t w e e n 75-100 mq and
labeled. Since the 2 a p p r o x i m a t e 1y equal segments of
tissue o r i g i n a t e d from a common culture, each pair was
referred to as sister calli. One sister callus was s u b c u l
tured onto fresh media supplemented with no NaCl and the
other sister callus onto 5 1 g mM <30 g /1) N a C 1. On a daily
basis far the first week, then on a weekly basis for an 12
additional weeks, calli selected for transfer because of
82their ability to p r o l i f e r at e on NaCl* and their c o r r e s p o n d
ing sister cultures from the control treatment were c o l
lected, Calli collected from N a C 1- s u p p 1emented medium and
from control medium were placed into 2 different petri
dishes (according to treatment) and mixed thoroughly. Two
gram samples from each treatment were removed. frozen in
liquid nitrogen, and lyophilized.
Sample p r e p a r a t i o n and e x t r a c t i o n of soluble proteins
One gram of each lyophilized sample was ground with a
Thomas motorized tissue homogenizer at full speed in 10
volumes of chilled acetone (- B O ° C ) in an ice bath, suction
filtered, and placed in a desiccator (under vacuum) until
all of the acetone had evaporated. Soluble proteins were
extracted with a Thomas motorized tissue homogenizer in ~n
icebath for 30 sec from 100 mg samples of the acetone powder
in 2 ml of lysis buffer c o n t aining 50 mM HEPES (pH 7.5 at q 0
C ) , mM DTT, 17 mM 2 - m e r c a p t o e t h a n o 1, 2 mM EDTA, and 1 mM
P M S F . The h o mogenized samples were transferred to 30 ml
Corex ce n t r i f u g e tubes and centr i f u g e d at 50,000 x G for 20
min at U 0 C. One hundred ul aliquots were removed from each
of the samples. The remaining supernatants were then c a r e
fully pipetted into individual screw top m i c r o c e n t r i f u g e
tubes, frozen in liquid nitrogen, and stored at -80° C for
03subsequent analysis.
Pr otein e s t i m a t i o n
The 100 ul aliquots p r e v iously removed from each of the
samples were analyzed for protein concentration. Total
soluble p r otein in each sample was determined by the Bio-Rad
pr o tein assay method. Bovine gamma g l o b u l i n was used to
qenerate a standard c a li br at io n curve for protein q u a n
tification. Absorb a n c e at 595 nm was read using a Bausch
and Lomb Spectronic PI spectrophotometer.
E l e c t r o phoretic co n d i t i o n s
Samples from the experimental calli containing 25 uq of
d is s o l v e d p r o tein in total volumes of 20 ul of buffer were
loaded into the we 11s of a 13’/. p o l y a c r y la mi de qel slab.
E l e c t r o p h o r e s i s was con d u c t e d according to the method
described by Laemmli < 1 2 > , however, a di s c o n t i n u o u s TRIS-
q l y cine buffer was used. Samples were e 1e c t rophoresed for
IP hours at 20 mAmps constant current in a refriqerated
chamber at 5° C. Molecular weight markers provided in W-SDS-
17 and M W - 5 D S - 2 0 Q p r otein molecular marker kits of the Sigma
Chemical Company were used. A total of 12 standards ranging
from 2 0 0,000 to 2 , 510 d a ltons were e 1ec t r o p h o r e s e d alonq
with the samples. Gels were then stained in 0.1257.
0 L
Coo m a s s i e RS50 for 0 hours* destained in 50*/. methyl alcohol
and 1OV. glacial acetic acid for 1 hr and then finally
cleared in 5 V. methyl alcohol and 77. glacial acetic acid
overnight. The gels were rinsed for one hour in distilled
water and then placed in a 0.17, solution of silver nitrate
for 30 min. Silver was developed in 3V. sodium carbonate and
0.05 7. f or ma l d e h y d e solution.
RESULTS
P r otein patterns of s a l t - adapted and control calli
Lanes 1 and 2 of the gel contain soluble proteins of
' P e n n e a g l e ’ and ' S e a s i d e ’ control calli* respectively
(Figure 1). Lanes 3 and ^ c o n tain the p ro t e i n s of salt-
adapted callus lines from ' P e n n e a g l e ’ . Lanes 5 and 6 c o n
tain the proteins of s a l t- a d a p t e d ' S e a s i d e ’. The first a p
parent d if fe r e n c e in p r otein banding occurred at a p
p ro x i m a t e l y 85.7-kd. There is a reduced p r otein band at this
location on the gel in both ' P e n n e a g l e ’ and ' S e a s i d e ’ salt-
adapted callus lines c om p a r e d with the corresp o n d i n q control
callus lines. There are several other p r otein differences
among cul t i v a r s and treatments* but the most strikinq d i f
ference was found in the protein bands welqhing a p
pr oximately 31.5-kd. It is at this molecular weiqht that
unique p ro t e i n s are synth e s i z e d in salt-adapted calli from
85
bath c u l tivars compared with their c o rresponding controls.
At ap p r o x i m a t e l y 19.1— kd and 18.2 kd» there appeared to be
cultivar p r otein differences. ' P e n n e a g l e ’ produced enhanced
levels of proteins at these molecular w e i q h t s . Since e q u i v
alent amounts of total p r otein were loaded into each lane,
it is assumed that diffe r e n c e s in the densities of the d i f
ferent bands represent differences in proteins. At a p
p ro x i m a t e l y 1B .9 - k d , calli from controls of both cultivars
produced enhanced levels of proteins compared to proteins
from their salt-adapted lines. ’S e a s i d e ’ produced increased
amounts of 18.2-kd proteins compared with both ’P e n n e a g l e ’
control and s a 1t -stressed lines and ' S e a s i d e ’ control
calli. There appeared to be additional d i fferences in the
lower molecular weight banding range.
Thirty days after being placed on control media, N a C 1 -
adapted calli from both cultivars failed to produce d e t e c
table p ol yp ep t id e banding at the 31.5-kd site on gels.
However, when placed back on N a C 1 -s u p p 1emented media their
growth was only slightly inhibited.
DISCUSS I ON
The results of this research corroborate some of the
findings of studies using tobacco < N i c o t i ana t ab ac u m s p p . >
liquid cell suspensions. E ri c k s o n and Alfinito (7), working
with N aC l - r e s i s t a n t tobacco cell suspensions, found that
there was enhanced p o l y p e p t i d e banding in the molecular
weight range of 32-kd and 20-kd. Singh et a l . <18) found in
creased levels of 21-kd, 19.5-kd, and 10-kd proteins in
N a C 1-adapted cell lines of cultured tobacco. Increased
levels of 19.1-kd and 18.2-kd proteins were detected in
' P e n n e a g l e ’ and 1B .5 — kd in ' S e a s i d e ’. However, whereas both
Singh et al. and Erickson a nd Alfinito ( 7 ) found a protein
unique to salt-adapted lines at 56-kdi the novel protein was
consistently found at 31.5-kd in both cul t i v a r s of creepinq
bentgrass. Another apparent differ e n c e was the decrease in
the 18.9-kd protein in NaCl -adapted Aqr os t is cultivars,
whereas Singh et al. (IB) reported an increase in protein of
that molecular weight range. Erickson and Alfinito (7)
found that when the N a C 1-adapted cell lines were transferred
to control medium, they lost their ability to grow in salt-
contai n i n g media and behaved like normal cells. There was a
complete loss in the p r o du c t i o n of both S O — kd and 3S-kd
p r o t e i n s after IS days (approximately 1 transfer period).
It took 2 to 3 passages for the 26-kd p r otein to become u n
detectable. Likewise, it took 30 days (one transfer period)
for the 31.5-kd salt-adapted bentgrass callus protein to b e
come undetectable. The presence of the unique 31.5-kd
pr otein in be n t g r a s s calli was detected after 2 weeks on
8 7
N a C l - s u pp le me nt ed media, and became more apparent with in
creasing levels of adaptation. It is, therefore, suqqested
that for b e n tgrass calli the 3 1 . 5 — k d protein is a salt
s t r e s s - 1 nduced protein. It may also be p os s i b l e that the
enhanced levels of 16.5-kd proteins play some role in the
a da pt a t i o n of 'Seaside' to N a C 1- s t r e s s .
LITERA T U R E CITED
Altschuler, M. and J.P. Mascarenhas. 1982. Heat shack
Mo 1 . Biol. 1 : 103-115.
Barnett, T., M. Altschuler, C.N. McDaniel, and J.P. M a s
carenhas. 1980. Heat shock induced p r o t e i n s in plant
cells. Deve. Gen. 1: 331- 39 0.
Baszczynski, C.L. and B.B. Walden. 1981. Regulation of
gene e x p r es si o n in c o r n (Zea mays L.) by heat shock.
Can. J. Biochem. 60: 560-579,
Bewley, J.D. and M.J. Oliver. 1983. Responses to a
c h anging environment at the molecular l e v e l : Does des-
s i c a tlo n modulate p r o t e i n synthesis at the t r a n s c r i p
tional or t r a n s 1 a t i o n a 1 level in a tolerant p l a n t 7 p.
195-169. In: D. Ran d a 11 (e d . > . Cur rent topics of plant
b i o c h e m i s t r y and physiology, Vol. 2. Univ. Missouri
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Brown, I.R. and S.J. Rush. 1989. Induction of a stress
p r otein in intact m a m m a l i a n organs after the intravenous
a d m i n i s t r a t i o n of sodium arsenite. Biochem. Biophys.
Res. 120: 150-155.
Cooper, P. and T.H.D. Ho. 1983. Heat shock proteins in
maize. Plant Physiol. 7 1: 215-222.
of heat shock in plants. Plant
09
7. Erickson* M.C. and S.H. Alfinito. 1989. Proteins
produced during salt stress in tobacco cell culture.
Plant P h y s i o l . 79: 506-509.
8. Fleck* 3., A. Durr, C. F n t s c h , T. Vernet , and L.
Hirth. 19 0 P . □ s m o t l c -shock ’s t r e s s - p r o t e in s ’ in
proto p l a s t s of M ic o t lana s y 1v e s t r i s . Plant 9cl . Let. 86:
159-165.
9. Hahn, G.M. and G.C. Li . 1982. Thermo to 1erance and heat
shock proteins in m a mm al ia n cells. Radiat. R e s . 92: 952-
957 .
10. K a n a b u s , 3 . » C.S. Pikaard, and J.H. Cherry. 1989. Hea t
shock proteins in tobacco cell s u s p e n s i o n during growth
cycle. Plant Physiol. 75: 639-699.
11. Key, J.L., C.V. Lin, and Y.M, Chen. 1981. Heat shock
p ro t e i n s of higher plants. Proc. Nat. Acad. S c l . U.S.A.
70: 3526-3531.
12. Laemmli, U.K.. 1970. Cleavage of structural proteins
during the assembly of the head b a c t e r i o p h a g e T-9. N a
ture. 227: 6B0-6B5.
13. Murashige, T. and F. Skoog. 1962. A revised medium for
rapid growth and b i o a s s a y s with tobacco tissue cultures.
Physiol. Plant. 15: 973-997.
1*+.
15.
1 6 .
17 .
18.
1 9 .
9 0
Nabors, li. W . 1983. Increasing the salt and drought
tolerance of crop plants. p. 165-189. In: D. Randall
Cur rent topics of plant bioc hemlstry and physiology,
Vol. E, Univ. Missouri Press. S t . Louis, MO.
Nabors, M.W. and T.A. Dykes. 1989. Tissue culture of
cereal c u lt iv a rs with increased salt, drought, and acid-
tolerance. p . 1E 1 - 139. In: Biotec h n o l o g y in i nt e r n a t l o n a 1
agricultural research. proce e d i n g s of the intei— center
seminar on international agricultural research (L A R C S )
and biotechnology. International Research Institute
Ph i 1 1 i p i n e s .
P o 1 j a k o f f - M a y b e r , A. 1900. Biochemical and p h y s io 1oqica 1
res p o n s e s of higher plants to salinity stress. Biosaline
Res. E 3: E95-E69.
Sachs, M., M. Freeling, and R. Qkimoto. 1980. The
anaerobic proteins of maize. Cell E 0 : 761-767.
Singh, N.K., A.K. Handa, P.M. Hasagawa, and R.A. Bres-
s a n . 1985. P r o t e i n s associated with adapta t i o n of c u l
tured tobacco cells to N a C 1. Plant Physiol. 79: 1E6-137.
Tal, M. 1983. S e l e c t i o n for stress tolerance. p.
961-988. In: D.A. Evans, LI .R . Sharp, P.V. Ammirato, Y.
Yamada, (eds.), Handbook of plant cell culture. Vol. 1.
M a c m i 1lan Pub 1 i sh i ng C o . , N . Y .
91
Fig. 1. One dimensional 5D5 Polyacrylamide gel slab. In a total volume of 20 ul, including sample buffert each lane contains 25 ug of soluble protein.Proteins from calli of ft . pa 1ust r is . 'Penneagle' and 'Seaside’ cultured at 0 mM NaCl are in lanes 1 and 2, respectively. Proteins from 'Penneagle’ calli selected over three generations on medium supplemented with 51^ mM NaCl are in lanes 3 and ^ . Proteins from calli selected over three generations on medium supplemented with 51^ mh NaCl are in lanes 5 and 6. Molecular weiqht standards are at the far left.
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APPENDIX
9 3
A N O V A TA B LE 1. P e r c e n t p a l u s t r 1s 'Penneagle' and 'Seaside' c a l l u sc u l t u r e s s e l e c t e d and t r a n s f e r r e d ove r t h r e e g e n e r a t i o n s .
SOURCE OFVARIATION DF 55 MS F-5TAT
Cu 1tivar (C ) 1 156. 3 156.3 1 .3S a 1t/Non (S ) 1 0556.3 0556.3 71.7**Generatn (G) 0 950. A A76.0 A .00 X S 1 000.0 000.0 1 .8C X G 0 535.0 067.6 P.PS X G 0 3A6.P 173. 1 1 .5C X S X G 0 1000.1 5A1 .0 A. 5*Error PA P866 .0 1 19 .ATotal 35 1 . Ae + OA
* S i g n i f i c a n t at .05** S i g n i f i c a n t at .01
95
ANOVA TABLE E?. Percent A . pa 1ust r i s 'Penneagle' and 'Seaside' cacultures with shoots. Resu1ts fcom three generat
SOURCE OFVAR 1A T ION___ DF S S MS F-STAT
Cultivar (C) 1 3.7 3.7 8.1**Sa 1t/Nan (S ) 1 31.9 31.9 70.8**Generatn (G) 2 6.5 3.3 7.3*C X 5 1 3.7 3.7 B. 1 **C X G 2 E. 1 1 . 1 2.3S X G a 6.5 3.3 7.3*C X S X G a a. i 1 .0 2.3Error 10.0 0.5Total 35 67.3
* Significant at .05* * Significant at .OtG
A N O V A T AB LE 3. P e r c e n t A^_ pa 1u s t r 1s ' P e n n e a g l e ’ a nd ' S e a s i d e ’ c al luc u l t u r e s w i th r oo ts . R e s u I t s f r o m t hr e e g e n e r a t 1o n s .
SOURCE OFVARIATION DF S5 MS F-STAT
C u 1t ivar (C > 1 831 -A 031 .9 27 . 0**Sa1t/Non (5) 1 3863.3 2862.3 93 . 1 **Generatn (G) 2 91 .6 95.B 1.5C X S 1 1100.1 1100.1 35.8**C X G 2 780.3 360. 1 11 .7**S X G 2 280.0 109.0 3.6C X S X G 2 179.2 87. 1 2.8Error 29 73B.0 30.8Total 35 6735.6
* S i g n i f i c a n t at .05** S i g n i f i c a n t at .01
97
ANOVA TABLE 9. Growth of A_ pa 1 ustr is 'Penneagle' and 'Seaside' callus cultures over three generations which had not increased in mass beyond original transfer size.
SOURCE OF VARIATION DF SS MS F-STA1
Cu1tlvar 1 92. B 92. B 0.9Sa1t/Non 1 1.6e + 09 1.6e+09 157.3##Gener a t n 2 337. 1 1SS.5 1 .7A X B 1 0.9 0.9 0.0A X C 3 90.9 20.2 0.2B X C 3 902 . 6 201 .3 2.1A X B X C 3 102.0 51.0 0.5Error 3h 2925. B 101.1Total 35 1 .9e+09
* S i g n i f i c a n t at .05## S i g n i f i c a n t at .01
98
ANOVA TABLE 5. Growth of A^ pa 1ustris 'Penneagle' and 'Seaside' callus cultures over three generations which increased in mass» but not double transfer size.
50URCE OFVAR 1 AT ION DF SS MS F-STA'
Caltlvar (C ) 1 33. 1 33. 1 0.6Salt/Non (S) 1 1 . 5e+0A I.5e+0A 873.8Generat n < G ) 8 688.5 311.8 5.5C X S 1 585.8 585.8 9.AC X G 8 86.0 13.0 0.2S X G 8 1 10.6 55.3 1 .0C X S X G 8 AA . A 82.8 0. AError 2A 1338.8 55.8Total 35 1 . 8e + 0A
» S i g n i f i c a n t at .05#* S i g n i f i c a n t at .01
99
ANOVA TABLE 6. Growth of A^ pa 1 ustris 'Penneaq1e ' and 'Seaside’ calluscultures over three generations which had doubled in mass from transfer size.
SOURCE OFVARIATION DF ss MS F-STAT
Cultjvar (C) 1 1 3 3 . 6 1 2 3 . 6 1 9 . 6 * *Sa11/Non (S ) 1 6.9e + 09 6. 9e + 09 7 6 2 5 . 0 * *Generatn (G) a 39.6 19.0 a . 3C X S i 305. 1 a e s . 1 33.0**C X G a 9.6 a . 9 0 . 3S X G a 153.9 80. u 9.1**C X S X G a 16.0 8.0 1 .0Error PR aoa.7 0.9Total 35 6.5e + 09
* S i g n i f i c a n t at .05** S i g n i f i c a n t at .01
100
ANOVA TABLE 7. Percent A_ pal ustr i s 'Penneagle' and 'Seaside calluscultures over three generations with 0 percent necrosis.
SOURCE OFVARIATION DF SS MS F-STAT
C u 11 1 var (C ) I 66.7 66.7 *r-in
Sa 11 /'Non ( 5 ) 1 6 . 2e + 04 6 .2e + 0A 52A3.1**Generatn (G > 2 ino 20.2 1 . C X S 1 1 A .6 1A .7 1 .3C X G 2 29A . 1 1A7.0 12.5**S X G 2 3.2 1 .6 0. 1C X S X G 2 212.7 1 06. A 9. 0**Error a u 202.7 U .BTotal 35 6.3e+OA
* Significant at .05*# Significant at .01
101
ANOVA TABLE B. Percent A_;_ pa 1 ustr i s 'Penneagle' and 'Seaside' callus cultures over three generations with 1-25 percent necros 1s .
SOURCE OFVARIAT ION DF SS MS F-STAT
C u 1tivar (C ) 1 2176.2 2176.2 55.6**S a 1t/Non (S ) 1 1 . 0e + 09 1.0e + 09 257.1*«Generatn (G) 2 192.5 96.2 2.5C X S 1 1 139 .6 1139.6 20. 0# *C X G 2 362.5 101 .2 9 .6*S X G 2 969.2 239 .6 *oin
C X S X G 2 1222.7 611.9 15.6**Error 29 939. A 39. 1To ta 1 35 1.7e*09
* S i g n i f i c a n t at .05** S i g n i f i c a n t at .U1
102
ANOVA TABLE 9. Percent A_ pa 1 us t r 1 s 'Penneagle' and 'Seaside' callus cultures over three generations with 26-50 percent necrosis.
SOURCE OF VARIAT ION DF SS MS F-STAT
C u 1t lvar 1 197B.B 1970.B 37.6* *Salt/Nan 1 1 .2e+09 1.2e+09 228.5*#Genera t n 2 236. 1 110.1 2.2A X B 1 1285.2 12B5.2 29 .9**A X C 2 675.0 337 .9 6. 2*B X C 2 16.9 B . 2 0.2A X B X C 2 155. 1 77.5 i 1“1 M
Error 29 1269.1 52.7Total 35 1 . 0P + O9
* S i g n i f i c a n t at .05** S i g n i f i c a n t at .01
103
ANOVA TABLE 10. Percent A pa 1ustr 1 s ’Penneagle’ andcultures over three generations with percent necrosis.
SOURCE OFVARIA T ION DF SS MS
Cultivar < C ) 1 32.3 32.3Salt/Non (S ) 1 1 1B2.2 1 102.2Generat n < G > 2 461 .3 230 .6C X S 1 31 .9 31.9C X G 2 216.9 100.5S X G 2 460 . 2 230. 1C X S X G 2 217.9 109.0Error 24 344 . 0 14.3Total 35 2946.7
'Seaside ’ call us greater than 50
F-STAT
£.302.5**16.1**2 . 2 7.6*
16.1**
* S i g n i f i c a n t at .05** S i g n i f i c a n t at .01
109
A N O V A TABLE 11. Percent A^ palustris 'Penneagle' and 'Seaside'regenerated calli over three generations producing shoots.
SOURCE OFVAR IAT ION DF S S M5 F-STAT
Cultivar (C ) 1 113.0 113.0 1 .35 a 1t/Non (5) 1 1 . 9e+09 1 . 9e+09 153.3*#Generatn (G) 2 093.5 921 .0 9.6#C X S 1 9312.1 9312.1 97.5#*X X G 2 909. 1 969 .5 5. 1*5 X G 2 O ru **4Oru 0.2C X S X G B 220.9 1 10.2 1.2Error 29 2100.0 90.0Total 35 2.3e+09
* S i g n i f i c a n t at .05## S i g n i f i c a n t at .01
105
ANOVA TABLE IE. Percent A_ pa 1 ustr is 'Penneagle' and 'Seaside'regenerated calli over three generations producing roots.
SOURCE OF VARIATION DF SS MS F-STAT
C u 11 i var 1 3i>A .2 36A. 2 A . 0Sa 11/Non 1 5A0.6 5A0.6 5.9*Genera tn a 60A .6 302.3 3.3A X B i IA12.5 1A 12 .5 15.5**A X C 2 5A7.2 273.6 3.0B X C 2 37A1.3 1070.7 20.5**A X B X C 2 A A . a 22. 1 0.2Error PA El 87.2 91.1Tota 1 35 9AA 1 .7
* S i g n i f i c a n t at .05** S i g n i f i c a n t at .01
VI TA
John Casper Hovanesian, the son of Luc iene and Ar-
shag H o v a n e s 1 a n , was born in New Britain, Co nnec ticut on
November 8, 1998. He was reard in Connecticut and M a s
sachusetts where he attended public schools for his
elementary and secondary education. In 1978, He
received a B.A. degree in History from the University of
Connecticut. In 1979, he received a B.A. degree in
M a r i n e Science from New England University (Saint F r a n
cis College). He was graduated the top ranking student
in the Department of Marine Science. His M.S. degree in
Biology was earned from the University of Southern M i s
sissippi in 1981. I n i 9 B 2 , he entered Louisiana State
Univer s i t y and anticipates receiving his Ph.D. degree in
H o r t i c u l t u r e with minor diciplines in Botany and Plant
P h y s i o l o g y in May, 1987.
1 06
DOCTORAL EXAMINATION ANI) DISSERTATION REPORT
C a n d i d a t e : John Casper Hovanesian
M a j o r Eield: Horticulture
T i t l e Of D i s s e r t a t i o n . IN VITRO REGENERATION AND PROTEIN CHANGES ASSOCIATED WITH TWO CULTIVARS OF Agrostis palustris Huds. CULTURED UNDER HIGH SODIUM CHLORIDE CONDITIONS
A pproved .
Major Professor and Chairman I k n m f r D . i t y s
I XT'Dean of the Graduate Ret tool
E X A M I N I N G C O M M I T T E E :
>-David H. Picha
( j , < T J ~ ^ - James F. Fontenot
0 <%/* Wk'-CV'—Blackmbn
t* t ; t. L L L- i' — -
Lowell E. Urbatschy L
Fogg
D a t e of E x a m i n a t i o n :
March 23, 1987