Radiation induced mutation rates and cyotlogical changes in plants orbited on Biosatellite 

Marimuthu, K. M.

Schairer, L. A.

Sparrow, A. H.

English

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Paper # BNL 13623 
4 I MASTER 
RADIOEIOLCCICAL STLTDIES OF FLANTS 
OXCTiGD m1 BIOSATZLIZTE I1 
L. A. Schairer, A. H. Sparrow and K. M. Merimuthu - P 
=-'lgy Departae~t, Brookhaven Xational Laboratory, Upton, NeuYoric 11973 
Beseerch sugpr ted i n  part by the U. S. Atomic Energy Com3ssion and in 
- 6j the Hational Aeroaautics a d  Space Administration (huchese Order 
To be preserited a t  the Space Biology Session of Worlccq Group V of fne 
C09a Yeet- in Prague, Czechoslovekia, 1day Xi-24, 1959, and t o  be published 
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Rediobiolo~iccl Studies of Plants Orbited i n  Ziosatell i te 11. 
L. A. Sckairer, A. H. Sparrow and K. M. Ehrimuthu. ~ i o l o e y  ~ c p s r t m . t  
Brookhiven Bational Laboratory, Uwn,  New York 11973 U.S.A. 
!W BiosateUite I1 'kadescentia e x p e r a t  probed the ef fects  of 
the space environment on spontaneous ana radietion-induced mutation ratts 
and on cytological chanees i n  Tradescantia clone 02. Tnirty-two young flw- 
ering plants were arranged in a p las t ic  housing with the roots inmersed in 
nutrient solution, were exposed t o  garam radiation from an on board 85 Stront lm 
source durlrii the tw-day orbi ta l  f l ight.  Unirradisted plants vere ilovn 
in a psc'kage i n  the spacecraft. behind a tuxs'm radiation shield and IdeutLcel 
no~%&t coxitrol peckages (with and without irradiation) uere mainhined a t  
'the launch s i te .  AYLr r e t r l w a l  of "Sle spacecraft near Havati, s q l e s  of 
root tip,  wary ard stmen tissues uere collected. These and the l!~te.ct 
t 
piants were ROM t o  Brookhaven for  observations on the folloving endplnts: . 
-tic Eutation, ce l l  size, loss  of reproductive inte&ty resulting in 
stunted s*-n hairs, pollen g r d n  mr ta l i t y ,  frequer.cy of micronuclei in 
pllw, disturbed mitotic spindle function and cdxmosazo aberrations. 
Aosrysis of &ta on scnatic mutation, c e l l  s ize  and chronosom aberraticw 
endpoints &are& no significant differences between f l ight  and no- 
s & * = ~ .  Bovnrer, po%n abortion, frequency of micronuclei in pollen and 
loss c f  reproductive integrity ( s b e n  h a i r  stunting) shnred increases 
essccia teavi th  wdghtlessness in irradiated materid. Root t i p  and micro- 
spore ce l l s  shoved effects of disturbed mitotic spindle function i n  orbited 
plants both v i th  and - . u t  irradiation. Clearly differences exist  between 
flight and nonfllght ma'terlal and the s&nificance and possible mechanisas 
fo r  these effects are be* studied in continrdng.nonNght tests.  ( ~ e s e s f i h  
supported by NASA (Puschase Order .~04-7) aad by USkEC.) 
? .$paceflight h c t o r s  acting alone o r  with ionizing radiation have 
ken &om t o  induce both synergistic and antagonistic effects a s  well as no 
significant diWerences, depending on the oremism and assay system used. 
ThZs paper discusses the current s ta tus  of the experiments v i th  the flowering 
plaat TreSescantie and su911arizes br ief ly  the  a n a l  results of the experiment 
vith tht bread w l d  Seurospora 3 in the Biosatell i te I1 mission. 
MAm- AND mmions 
B e  Tradescentia Wer inen t  on board Biosatell i te II(') was designed 
to deternine the effects of  weightlessness and other spacecraft envi-ental 
conditions on spontaneous and radiation-induced mutation rates and on varioue 
cytolagical chanses in a s p c i a l  clone of the hieher plant, Tredescentia. 
This h y M d  clone of Tradescentis has e chromosome number of 12; It is heter- 
o m  fo r flower color; end it has a hleh mutation ra te  a t  the locus deter- 
Piniag fiover color (storable i n  both petal and s t m n  hai r  cells). During 
the h5 br BiosateUite fl lght,  32 young plants were arranged in  a p las t ic  
boas* so tha t  the flower buds were exposed to about 220 R of gama rays 
EWLLE the mots, luimersed i n  nutrient, were exposed t o  known radiation levels 
about 125 t o  285 R. 'Phirty-tvo f l l gh t  control plants were noun i n  an 
identical  package in the spacecraft behind a tungsten radiation shield and 
da%lar =flown control packages (With and vithout irradintion) were Mxta ined  
at the Istmch site. The internal spacecraft environment was rigorously con- 
+rolkd snd Mnditioas moni toredthro~hout  the fllght. The temperatures were 
reiotsiaed between 65 and 70' F i n  the  Tradescsntia packages, the atmospheres 
hcld at about 5% relative humidity a d  the gravitational force a t  about 10-5n 
during the irradiation phase of the fl ight,  Telemetered and on-board records 
vere made of vibration, shock, acceleration, etc. for  use in 6ubcequmt kst6. 
Inrmediately a f t e r  retrieval of  .the spacecrafi near mil, root-tip, o v e y  as6 
stamen tissues were collected. These and the in tac t  plants w r e  ZloMl to I 
BrooWlaven for  observations on the following end points: r;OJiatic mutation, I 
c e l l  size, loss  of reproductive in teer i ty  ( c e z  &nth and s tun t iw  in stamen I 
ha i r  growth), pollen grain mortality, spindle malfunction and chromosome aber- 
I, 
. rations. :, 
p l l o v h g  prellm5nary analysis of the engineering and biol.o&zal I 
data, s e v h  p o s t - f l w t  Eartb-based exye&ents were performed under con I 
t ions as c b s e  es possible t o  those o f t h e  flight. These tests, run i n  
6- N&t hrdware, vere designed to establish or recheck base-ll& data an 
.the effeets of irrar!lat2on, vibration, b i o c ~ 2 t i b i l i t y  of the spacecrafi, etc 
& a a a t i c  mutation zates were determlnea 'for pi-t changes dxrm 
1 1 .  
the dcPinmt: blue t o  pink in both petals and stamen &irs and to colorless in 
Sn ~ e n e r a l  
only the &a& hairs (see Table 1). The mutatian dts/showed the expected 
increase dw, I% the gezrm exposure but the f l lght  end nonfllght sevqles 
shoved no &?ereaces attr ibutable to flight factors.J Although the peak 'I 
thaq expected .,I 
mutatLon rab fo r  the pink stme; ha i r  ce l l s  vas h r  / in the flAght a  
l i t t l e  s&Dticance vas attached to this reduced rate f-U6e a sidlar 
uss observed in a ' p o s t u h t  test. A l l  unirradiated &ts had conslatent 
low mu'Jltion ra tes  and shoved no differences .between f l i gh t  and nonnight 
The frequency of dbmrf and giant ce l l s  increased following g m a  
urposye, but there were no significant differences i n  the peak yalbes of 
f l i gh t  and nonf)leht Ixadia ted samples. m b -  
me loss  o r  pa r t i a l  loss  of reproductive integrity of terminal o r  
subternlnal stanen hai r  ce l l s  resul ts  i n  a short o r  stunted hair. Hair 
lengths norselly vary soneuhat, but the incidence of stunted hairs increases 
vith increas- exposure and postirradiation tine. The percentage of  stunted 
hatrs generally was higher in  the flight-irradiated than i n  nonfli~ht-irradi-  
atat samples aad average values for  the period between 9 knd 17 deys a f t e r  
a ~ e v a l  ere given in  Table 1. Ibny more very short hairs (5  ce l ls  or  less)  
i observed i n  the f l i eh t  sample then i n  the coaparable non-fl1eh"u sa?ple, 
indicating that  &&e same radiation exposux-e produced a greater deleterious 
ef fect  on c e l l  dv i s ion  in the biosate l l i te  than in  the EarL&-based plants. 
Mature pollen wes collected daily, end, a f t e r  staining with cotton 
blue, the srcenteges  of aborted pollen were detennined for a l l  four t rea t -  
ments (see TaSle 1). It should be emphasized that t h i s  clcne of Tredescwtia 
S.6 --haracterized by an abnormally high re te  of spontaneous pollen abortLon 
9 9 (average ccmfrol levels were 3 and 9x5 for f l ight  and g r o u ~ a  cant&, 
respectively). m e  data, however, indicate that  there was generally a 3 i ~ h t r  
abortion r a t e  i n  the flight-radiation sample than i n  the nonflight-radiation 
. semple v i th  sone points being s i p l f i c a n t l y  different a t  about the one percent 
level. These resul ts  were further supported by the data on the frequency of 
mlcmauclei observed a f t e r  staining with propiono cernine (see Table 1). 
' n The 2-:old increase on certain days in frequency of micronuclei i n  
the irradiated f l ight  sample reflects an enhanced effect of f l ight  fac 
and irradXation on cel ls  treated during the sensitive stages of meiosl 
(14-18 day6 before anthesis) . 
Collections of young microcpores w-de a f t e r  atat shoved an 
pecteay high death ra te  in both f l ight  sanples. All o f t h e  microspores 
buds treated during microspore mitosis had an abortion ra te  of mare than 9%. 
Subsequent Earth-based t e s t s  in the f l i gh t  vebicle (301) produced similar 
microspore death to ths t  of the oraited a t e r i a l .  This effect nay therefore 
be due to s m e  enviromental variable i n  the vehicle rather then t o  N g h t  
conditions. PrelJalnery experirrnt.s indicate that  ethylene nay be one of ll 
the rectors Con'lributing.to tiis c e l l  deeth. 
I n  the surviving fraction ((55) of the microspores, abnonsalltj 
rea?ect4q disturbances i n  the spindle mec-m vere observed a t  a nuch 
higher trepuency in the f-ht control. (27.95) than In the nonflight control I 
(0.1€$). 1 
dence of a disturbance i n  the spindle mechanism also was s e a  In 
material in the f o i  of multinucleate ce l l s  and in pecu- 
nuclei. Ta3re 1 gives the percentage of such ce l l s  in nonflight 
riels. The percentages are mall when expressed a s  a per cent 
but would be much larger i f  only ce l l s  a t  metapbase t o  
/ steges were scored. However, it is apparent that  not all ce l l s  
evidence of  a spindle inhibition or  malfunction. 
I 
@s%osee aberrations were scored a t  netaphase i n  root-tlp ceUd in 
BU treatments. W s i p l f i c a n t  differences were found between 
I U 
I 
data horn the respective control and irradiated flight and nonflight sam?les. 
Other post-flight tes ts  included studies of the effects of vibration 
and the clinostet on the various endpoints used. Work with the zlinostat 
(2 revoiutions per ninute) with and without irradiation can be' quickly sum- 
m8xtze.i ?s ha- had no effect or: any of the endpoints scored. 'Flight-simulated 
vibration tests  on both the entire capsule and individual packwes also failed 
to &ow acy sig-ificmt effect on the Tredescantia plants. 
The results of the Biosstellite I1 and Gemini XI PTeurospo?a expr i -  
mats of de Serres, e t  el. of tiie Oak Ri&e Lational Laboratory ( 2 9 3 )  w i l l  be 
sumaized o.nly briefly here since this  work is essentiany conplete arid has 
bee21 repozteQ a t  tiie l a s t  COS?A.. :<eeting (6:ay, 1968). 
. 
Dose respnsq curvec were nade from data on cellular Inactivation 
an& the Ssduction of mutetiors (recessive lethal mutations occurring over the 
eatire genme as vel l  as a t  two specific loci). , In both the Biosatellite I1 
mid GerLni XI experiments, using samples collected on millipore f i l ters ,  there 
uere no effects of spaceflight alone or with ionizing radiation on inactivation 
or mutation-induction. However, whea the Neurospora samples were f h  in
smpension i n  the Gemini X I  experime2t the trradiated ssnples showed a higher 
survival rate and significantly lowar rate of s-eiric locus mutations th s 
+be nanflight samples. 
COBiCKJsIQ?S 
Thus there are some effects on spantaneous and radiobiolo~cal  events 
in TnCescantia plants w'dch may be attributed t o  orbital end simulated f l ight  
-ts. These effects are rather smU but significant and aqpear t o  be 
related to  effects on nornal ce l l  division and cel l  s w i v e l .  2ud bkstini: 11 
i 
and dcrospore death are probably due to s o x  vehicle w i r o ~ ~ e n t a l  cffccts 1~ 
anr?.not flight factors. High rates of pollen abortion, s t u n  hair stunting I1 
and formn"ion of micronuclei are unique t o  the irradiated, orbited sa~jjk and 
reflect an enhanced interaction between radiation and wei&tleceness du- I 
the rare sensitive stages ( meiosis aid mitosis).. Nuclear misoKentation 
associated with mitotic spindle nalfmction i n  pollen and root ti? cells were 
jncreased significantly in  bot'a irradiated end tmirradiated flight sez@les as 
c-zed viva shuleted flight se3lples. Tnese la t t e r  ai.lormalities were similar 
to those observed by the S o i d  ocientists (495) and are probably at t f iWdble I 
to weightlessness elone but my conceivabl~ be due to wei&htlessness preceded 1 
enSi!or follosred 5y vibration b r  other dyn&c factors associated vlth orbital I 
flight. Clearly differences exist between ?light and nonfli&%t sasples Wt I 
v i t h  and vfthout 1,madiation. The signiiYcence and possible nechanisn;s for I 
these e f f s d s  as vell as develo~zntal  studies h o l m  or5i';ed mgaspore I 
tissues are being investtgated in continui3g nonflight tests. I 
fn the Heu;oqora ~ W n t  l2?e genetic effect Of radiation on 
nmdividhg end ~ b o l i c a l l y  inective cells was identical for  flight a ~ i .  
non-t s e q l e s  (the sasples collected on millipore f i l ters) .  Mfferences 
- 
were found'oetveen 4i&t and nonflight sasples vhen atabol ical ly  active cells 
(the san@es in suspensim) were used. 
1 1. Spmro-ir, A. B., L. A. Schairer and X. 16. Marimuthu. 1958. Genetic and 
cytologic studies of Tredescantic irradiated duin(l orbi ta l  
fli&t. Bioscience e. 582-590. 
1 2. deSerres, F. J. and B. B. Yebber. 1965. nUCtiv8tion and mutation- 
induction i n  Reurospora resulting horn combined rediation and 
veightlessness on tine Biosstt=?lite I1 missioa. Bioscienca 18: 
590-595- 
/ 3. deSerres, F. J., I. R. Killer ,  D. B. Smith, S. Kondo, and 14. A. Bender. 
s 6 9 .  T5e Gemini-XI S-4 spaceflight radiation interaction 
experkent 11, Aneljrsis of survival levels and forward-~uutation 
frequencies in  Xeutvswra crassa. Radiation Res. (in press). 
4. Delone, B. L., V. F. BykOvskiy, V. V. Antipov, G. P. Parfenov, V. G. 
Vysotskiy and B. A. Rudneva. 1964. Effect of space factors on 
the  microspores of Tradescantia paludosa i n  the  Vostok 5 ar.d 
Vostok 6 spacecreft. NASA 'IT F-8896. Natl. Aeronautics and 
Space Administration, Washington, D. C. ( ~ r a n s l .  from ~ u s s i a n )  .
17 p. Oz%ginal publ. i n  Kosmicheskiye u. 5 320-329 (196L). 
1 5. m e ,  It. L., A. 8. -ova, E. I. Yorozova, V. V. Anmunov and G. P. 
I Parfenov. 1963. Effects of space f l i gh t  of the sputnik Cosmos-llO 
. . 
-9- 
Table 1. S u m c r y  of Data for  Verious 'Iredcscant:~ Endrn:ntc f r o m  a:osatcllite 
Flight (301) Konfli&t (201) 
Endzoints Irrad. Unirrad. Irrad. Unirrad. '1 
I 
, 
1 3 S?.kCZ ?LIGST I3FDCTS 
>:utations (so.mtic) 
~ i c k / p e t a l  20.6 2 2 . 9  1.4 20 .2  15.4 2 2 . 9  1.1 20.3  
~ink/100 s t o z n  hai rs  4 . 2 2 0 . 8  0 .320 .2  8 . 2 ~ 1 . 3  0 .220 .1  
~ o l o r l e s s / l ~ ; r  st~-:en hairs 7.6 5 1.6 0.1 + 0.1 7.3 5 - 1  0.3 + 0 - 1  
. -- - ----- -- - 
Dnr f  cells/lGO hairs 12 .321 .4  3 . 2 5 0 . 6  15.451.7  3 . 0 ~ 0 . 7  
Giant c e ~ s / 1 0 0  hairs 5.18 2 0.67 4.51 2 0.70 
- - -  - -.-. 
~ttrar.osor.e a b e r . / c e ~  ( R O O ~ ~ )  0.53.~0.091 0.04 20.021 -0 .48~0 .096  0.02 ~ 0 . 0 1  
E?;.w!CE3 ~ 5 ' s  
Loss of repod. i n t e , l t y  
(stuntirg)/lCO heirs 27.0 22 .6  10.3 21 .5  12.9 21.7 8.2 f 1.2 
i a c r o n ~ c l e i / l ~ ~  ce?ls 
Pollen 21.1 a.9, 
-- -- - - - - -- 
1 
- I 
~ i c o s p o r e  &at- $ buds I* 16.@ 33 .3  
M s t u r s d  spindles ($cells) 
0.55 + 0.08 0.25 + 0.0: 0.06 2 0.03 0 I 


Radiobiological studies of plants orbited in biosatellite 2

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