The effect of benzene hexachloride isomers and certain enzyme inhibitors on cell division in plant roots

77 Pages·05.283 MB·English

by  Hopkins, Homer Thawley

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THE EFFECT Of BENZEHK HBXACHLORXDE ISOMERS AMD CEKTAIH
BHZ1US INHIBITORS OH CELL DIVISION IN PLANT ROOTS
by
Homer Thawley Hopkins, Jr.
Thesis submitted to the faculty of the Graduate School
of the University of Maryland in partial
fulfilleent of the requirements for the
degree of Doctor of Philosophy
19$1
UMI Number: DP70403
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vi
Acknowledgementst The writer wishes to express his appreciation to
flrs* G&uch and Dugger for their aid* particularly in the preparation
of the thesisj to Dr. s. B. Hendricks for his guidance and interest
in the problem} and# to Dr# A* W. Specht for helpful counsel on certain
technical aspects*
±63839
vil
TABLE OF COMTFJITS
rage
Introduction .................................. .......... . I '
Review of Literature 3
Description ofM aterials and Experimental Methods 10
Rooting Medium 12
Plant Material .... 12
Cultural Methods ........................... .. • 13
Experimental Techniques 13
Chemical Methods »«••»»•••«•*»••••••••»•*••••••••• 16
Results ..... 17
Preliminary Data .........•••••••«••••••••« 17
Screening Experiments •••••••••«•••••••••••••••••• 17
Cell Counting Technique • ••...... 18
Dosage Response Experiments ••••••«•*••••»*•«•*••• 30
Time Course Experiments •••••*••*••••»•••••••••••• 3h
Transfer Experiments .... 37
Effect of Various Chemicals on Cell Division ••••• h2
Antagonisms .......... ...*••......• Ii3
Inorganic and Labile Phosphate Analyses 146
Discussion of Results and Conclusions ••••••••••••»••••••••*•» h9
Summary 56
Literature Cited .... 58
vi.il
LIST Of TABLES
Table Page
1 .Description of benzene hex&chloride (1,2,3*14,5,6 hexa-
chlorocyclohexane) isomers.......................... 11
2 Description of enzyme inhibitors and pH of solutions
used ........................... 15
3a Effect of seed age on cell numbers in excised Cucurbita
pepo seedling root tips in response to benzene 'gamma
hexachloride in a 1*8 hour e x p e r i m e n t * 19
3b Effect of pre-aeration of tap water for 2h hours on cell
numbers in excised root tips, and root lengths of
Cucurbita pepos eedlings 19
3c Effect of presence of Tween-20, 0.08$ in tap water,
on cell numbers in excised root tips and on root
lengths of Cucurbita pepo seedlings ....... 20
h Results of screening benzene hexachloride isomers on
five different plant species; cell numbers in excised
root tips and lengths of seedling roots in a ltd hour
experiment 21, 22
5 Cell counting technique; values for total cells and
non-vacuolated cells obtained in successive samplings
from two different sample bottles; untreated excised
roots of Cucurbita pepo 23
6 Cell counting technique; values obtained for total
cells and non-vacuolated cells in successive samplings
from two different sample bottles; EHG treated excised
roots of Cucurbita pepo ......... 2i+
7 Average root lengths of fifty seedling! selected at
random from twenty-five separate experiments, and co­
efficient of variability for treated and untreated
seedlings; Cucurbita pepo 26
8 Variation in cell numbers between fifty treated and un­
treated samples selected at random from 25 experiments,
total and nan-vacuolated cells per root of Cucurbita
pepo .....*..... .......o........... 27
9 frequency distribution of total cells, non-vacuolated
cells, and root lengths; random selection of data from
25 different experiments, treated and untreated seedling
excised root tips of Cucurbita pepo....... 28
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LIST 01 TABLES (Continued)
•"'■■rtWiWiii'w 1 .'*i i'ii-..“vf-iis (WW!H»p»4p
Table Page
10 Dosage response data for Cucurbita pepo-, using benzene
gama hexachloride in a hour experiment, showing
average number of total cells and non-vacuolated cells
per root and average root lengths • •»........ 31
11 Dosage response data for Cucurbita pepo, using benzene
delta hexachloride in the'rootSg"'"'S'cS.'iaj duration of
experiment 1*8 hours ................................. 32
12 Dosage response data for Carthamus tinctorius and
Helianthus arrnus using benzene gamma hexachloride in a
Uo hour experiment, showing average numbers of total
and non-vacuolated cells per root and average root
lengths ........... . 33
13 Tiiae course of cell division and cell elongation of
roots, average cell numbers and root lengths asso­
ciated with various benzene gamma hexachloride treat­
ments in a JUS hour experimentj Cucurbita pepo ........ 35
Ih Indices of cell division and cell elongation from the
time course experiment, calculated from the ratio be­
tween non-vacuolated and vacuolated cells in the former
case and in the latter case from the ratio between
length of excised root tips and number of vacuolated
cells: Cucurbita pepo ...... 36
1!$ Cell numbers and root lengths as affected by contact
time and various concentrations of benzene gamma hexa-
chloride, Cucurbita pepo seedlingsj duration of
experiment, no hours •.. ...... * 38
16 Indices of cell elongation and cell division calculated
from the transfer experimentj Gucurbita pepo 39
17 Indices of cell division and cell elongation in roots
of treated and untreated Cucurbita pepo seedlings;
fifty randomly selected samples from twenty-five ex­
periments ........ ...... ........................... la
18 Enzyme inhibitors and the concentrations which caused
inhibitory effects on cell division and root growth
of Cucurbita pepo— criterion of effects being index
rmt A**"*." .whm1 m
of cell division ......... ......... . hh
19 Overall root lengths, cell division index, and per­
centage protection exhibited by various compounds
against inhibitory effects of benzene gamma and delta
hexachloride on roots of Gucurbita pepo •••••••••••«••• 16
X
LIST QF TABLES (Continued)
Table Page
20 Failure of glucose to protect against the onaitotic
and c-tuaor reaction in the presence of benzene gamma
hexachloride at a concentration of 5>.7 x 10"“%. ....... hi
21 Changes in concentration of inorganic and labile phos­
phate in Cucdrblta pepo roots after various contact times
with benzene gamma hexachloride and 2,U-dinitrophenol .. hi
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LIST OF FIGURES
Figure Page
1 Comparison of ©ell division index for treated and uiv
treated roots of Cucurbita pepo, in a JUS hour experi-
'3U
ment ................................
2 Comparison of elongation index for treated and untreated
roots of Cucurbita pepo, in a 18 hour experiment •••••• 3Ub
3 Change in the inorganic phosphate content of squash roots
with time of contact in media treated with benzene gamma
hexachloride and 2,l;~dinitrophenol U7a
IMTJtOlJyCTlJM
The purpose of this study is to present and discuss certain data ob­
tained by a technique permitting evaluation, in vivo, of (1) the effect
of benzene hexachloride isomers on cell division and cell elongation of
plant roots, and (2) the metabolic antagonist of these effects by spe­
cific enzyme inhibitors or other chemical antagonists*
One of the most unique properties of protoplasm is that of revers­
ible gelation and eolation* A gel offers a logical structure with plenty
of surface© for enzymic and adsorptive processes upon which protoplasmic
function depends* Cellular function requires a high state of organiza­
tion of the protoplasm, and in order to maintain this highly organised
state, the cell must perform work* Energy for this work must coca© from
metabolic chain reactions catalysed by the intact ensyme systems of th©
cell* Covering the protoplasm of a cell is a surface film presumably
consisting of a palisade-like structure of lipo-protein complexes so ari
ranged that the protein part of the complex is directed to the exterior*
This film or protoplasmic membrane is regarded as being constantly re­
newed and formed. As long as the surface film of protoplasm is & con­
tinuous envelope, and its composition stays within certain bounds, the
integrity of the protoplast may be .taintained*
Benzene hex&chloria© possesses certain properties characteristic
of typical narcotic compounds. Two of these properties may be mentioned,
namely*} lack of polar groups, and limited solubility in water. Owing to
the lack of polar groups, it would be expected that benzene hexachloride
molecules would accumulate preferentially in the lipoid phase boundaries
of protoplasm, following such accumulation, specific effects on cellular
2
function, in vivo, could occur* Those specific effects suggested points
of departure for an objective study of benzene hexachloride ami related
coapounds*