Cytological consequences of proembryo irradiation

Radiation Botany, 1969, Vol. 9, pp. 269 to 282. Pergamon Press. Printed in Great Britain.

CYTOLOGICAL CONSEQUENCES OF PROEMBRYO I R R A D I A T I O N * L. W. M E I I I ~ I . E a n d llt. P. M E l l I C I , E Department of Botany and Plant Pathology, and Biology Research Center, Michigan State University, East Lansing (Received 22 November 1968) Abstract--Cytological responses (including effects on embryo size and cell number, changes in mitotic cycle time, and production of chromosome aberrations) exhibited by early and midproembryos of barley (Hordeum distichum cv Hannchen) following acute X-irradiation (450 R) were examined in the hope of finding explanations for some of the striking differences in radiosensitivity between these stages (e.g., embryo survival and anomalous organogenesis). In irradiated early proembryos strong diplontic selection appears to operate against cells with gross cytological aberrations, which, because of the small number of ceils present, makes embryo abortion common. Surviving embryos are mostly ones escaping major damage or reconstituted from one or a few undamaged ceils. These embryos, therefore , are relatively homogeneous for absence of gross genetic deficiencies during what seems to be the 'critical period' for production of organ abnormalities, i.e., just prior to or at the very onset of differentiation stages in embryogeny. Presumably this accounts for organ anomalies being so rare after irradiation of early proembryos. Irradiated mid-proembryos, by contrast, tolerate the continued presence of cells with gross cytological aberrations, and must be, therefore, highly heterogeneous, genetically. Intercellular incompatibilities engendered by such heterogeneity during the 'critical period' are postulated to be responsible for the high frequency of anomalous organ development associated with mid-proembryo irradiation. Evidently the presence of gross cytological aberrations during actual organ differentiation does not produce abnormality nor does their presence per se result in growth retardation or disturbance of mean mitotic cycle time. Partitioning of cellular components may account for the changes in genetic partiteness manifest during proembryo development. ll~suJm~---Dans l'espoir d'expliquer certaines difffrences importantes de radiosensibilit6 entre difffrents stades (par exemple la survie de l'embryon et les anomalies de l'organogen~se), on a examin6 les r6ponses cytologiques (y compris les effets sur la taille de l'embryon et le nombre de ses cellules, les changements de dur6e du cycle mitotique et la production d'aberrations des chromosomes)-montr6es par des embryons d'orge (Hordeum distichum cv. Hannchen) prdcoces et moyens, h la suite d'une irradiation aigufi (45 R). Dans les pro-embryons irradi6s pr~cocement une forte s61ection diplontique parait bien s'effeetuer eontre les cellules qui poss6dent des aberrations cytologiques majeures et qui en raison du petit nombre de cellules font commun6ment avorter l'embryon. Les embryons qui survivent sont surtout ceux qui 6chappent ~t ce dommage majeur ou qui se reconstituent ~t partir de quelques cellules non 16s6es. Ces embryons sont d6s lors relativement homog~nes quant ~t l'absence de ddticienees gdn6tiques majeures pendant ce qui paratt ~tre la 'p6riode critique' en ce qui concerne la production d'anomalies organiques c'est-~-dire la p6riode qui se situe juste avant ou a l'extrgme dgbut de la diffdrenciation des stades embryonnaires. Cela explique probablement que les anomalies *Research carried out under the auspices of the U.S. Atomic Energy Commission. Tech. Rpt. COO-1400-20. 269

270

L. W. MERICLE and R. P. MERICLE organiques soient si rares apr~s irradiation des tout jeunes pro-embryons. Par contre, les proembryons moyens tol~rent apr~s irradiation la pr6sence continuelle de cellules avec des aberrations chromosomiques majeures et doivent alors ~tre fortement hft6rog~nes du point de rue gdn6tique. On postule que les incompatibilit~s intercellulaires engendr6es par cette h6tdrog6n6it6 au cours de la "p6riode critique" sont responsables de la frfquence 61ev6e d'anomalies du d6veloppement des organes associ6e 5, l'irradiation de l'embryon de taille moyenne. De toute 6vidence, la pr6senee d'aberrations cytologiques majeures pendant la p6riode de diff6renciation effective de l'organe ne produit pas d'anomalie et n'engendre d'ailleurs per se aucun retard de croissance ni anomalie du temps moyen du cycle mitotique. La r6partition des composantes cellulaires peut expliquer les changements gdn6tiques qui se manifestent pendant le d6veloppement du pro-embryon.

Zusamrnenfassung--Bei Proembryonen yon Gerste (Hordeum distichum cv Hannchen) im fri)heren und mittleren Entwicklungsstadium wurden die cytologischen Reaktionen nach Behandlung mit R6ntgenstrahlen (450 R) untersucht, um Erkl/irungen zu finden ftir einige der auffallenden Unterschiede zwischen diesen Stadien hinsichtlich der Radiosensibilitiit (z.B. ~berlebensrate der Embryonen und anomale Organogenese). Die Untersuchung umfasste Auswirkungen auf Embryogr6sse und Zahl der Zellen, Ver~inderungen der Dauer des Mitose-Zyklus und Bildung von Chromosomen-Aberrationen. Bei Bestrahlung yon Proembryonen in der frtihen Phase schien eine starke diplontische Selektion gegen Zellen mit starken cytologischen Aberrationen wirksam zu sein. Diese Selektion bedingt ein allgemeines Absterben der Embryonen, da die Zahl der Zellen gering ist. Es ~berleben meist solche Embryonen, die nicht stark gesch/idigt wurden oder aus einer oder wenigen unbeszhiidigten Zellen regenerierten. Diese Embryonen sind daher relativ homogen, insofern sie keine grossen genetischen Defekte wahrend der Zelt aufweisen, die die "kritische Periode" ftir die Bildung yon Organ-Anomalien zu sein scheint, d.h. in der Zeit vor oder zu Beginn der Differenzierungsstadien im Embryo. In bestrahlten Proembryonen der friihen Entwicklungsphase treten vermutlich deshalb Organ-Anomalien so selten auf. Bestrahlte Proembryonen der mittleren Entwicklungsphase dagegen tolerieren das dauernde Vorhandensein yon Zellen mit starken cytologischen Aberrationen und miissen daher genetisch stark heterogen sein. Es wird postuliert, dass interzellulAre Inkompatibilit~iten, hervorgerufen durch solche Heterogenit~it w~ihrend der 'kritischen Periode', ftir die grosse Hiiufigkeit anomaler Organentwicklungen verantwortlich sind, wie sie bei bestrahlten Proembryonen der mittleren Phase auftritt. Das Vorhandensein starker cytologischer Aberrationen wiihrend des Differenzierungsvorgangcs der Organe bringt offensichtlich nicht die Anomalien hervor, noch resultiert ihr Vorhandensein an sich in einer Wachstumsverz6gerung oder St6rung des zeitlichen Ablaufes des mitotischen Zyklus. Die Aufteilung der Zellkomponenten kann eventuell verantwortlich gemacht werden ftir Aufiinderungen der Aufteilung des genetischen Materials, die sich wiihrend der Entwicklung des Proembryos manifestiert. ..

V

INTRODUCTION MARKED changes in radiosensitivity take place during barley embryogeny, especially during the proembryo period of development. The effects of acute irradiation applied at specific embryonic stages have been investigated in our laboratory over recent years from a variety of genetic, histological, and physiological standpoints.C2,5,e,9,1620) We have long recognized the need, however, for additional cytological information in the expectation that such information would help to explain some of our previously observed effects

,

on embryo survival, mutation rates, organogenesis, and cellular components. To this end, we have been conducting a series of experiments during the past two years to obtain more definitive data on the cytological responses of irradiated proembryos in terms of their subsequent growth in size and cell number, mitotic cycle time, and production of chromosomal aberrations. I n the present report major emphasis is placed upor~ a comparison between embryos irradiated at early vs. mid-proembryo stages since these two periods in embryogeny

CYTOLOGICAL CONSEQUENCES OF PROEMBRYO I R R A D I A T I O N exhibit the most consistent and striking differences in degree of radiosensitivity and patterns of response.(~8)

M A T E R I A L S A N D 1VIETHODS

Proembryos of Hordeum distichum ev Hannchen (C.I. 531), developing in situ on the parent plants, were irradiated with a single, acute exposure of 450 R X-rays delivered at a rate of 170 R/rain. Physical factors for irradiation were as follows: 200 kVp, 15 mA, and an inherent filtration of 3 mm A1. Except during the few minutes of irradiation, the plants were maintained in a controlled environment growth chamber operated at 22°C with a ~4000 ft-c light intensity during a 16-hr day and at 13°C during an 8-hr night. At the time of irradiation and selected intervals thereafter (from 1 to 15 days post-radiation), embryos were sampled for cytological examination from irradiated and control materials of similar age and the same initial embryonic stage. Embryo sizes and cell numbers were determined from both sectioned and squashed Feulgen-stained preparations; proportion of cells in division and frequencies of mitotic bridges, chromosome fragments, and micronuclei, also from squashed Feulgen preparations; and stages of embryonic development, from sectioned preparations and dissected materials. Volumes of proembryos and early differentiating embryos were calculated from length and width measurements of medial longitudinal radial sections through the ovary, assuming a prolate spheroid configuration for the embryo.

®0 a~ zygote

b

EARLY PROEMBR¥0S

I

early e

late f

MID-PROEMBRYOS

FIo. I. Stages in normal barley embryogeny signified by the designations 'early proembryos' (embryonic stages a through b, I-2 cells) and 'mid-proembryos' (embryonic stages e-~ ~ 30-600 cells) as used in tbe text.

271

Designations of embryonic stages in this presentation follow those used in earlier studies (3,16) wherein the proembryo period of development is divided into 7 stages (a-g) and the period of organ differentiation into 6 stages (I-6), with the last further subdivided into 3 stages of maturation (6a-6c). For discussion purposes here, embryos in stages a and b (1-2 cells) will be referred to as early proembryos, and ones in stages e a n d f (30-600 cells) as mid-proembryos, Fig. 1.

RESULTS

Normal emblyogeny T h r o u g h o u t the p r o e m b r y o period and into the early stages of differentiation, cell n u m b e r in non-irradiated control embryos increases exponentially with a m e a n mitotic cycle time of ,-, 12 hr under the environmental conditions used in this study (Fig. 2). Thus it requires a m e a n of 10 cell divisions and nearly 5 days time for the barley embryo to progress from the 1-celled zygote to the end of the p r o e m b r y o period as typified by an embryo comprised of ,,~ 1000 cells. O f course, not all cells of the embryo divide with the same fi'equency during this time. Cells in the basal region, for example, undergo fewer mitoses than those situated more apically, the latter eventually giving rise to shoot and root regions in the differentiating embryo.(S~ I n very y o u n g proembryos, most if not all of the cells m a y be in some stage of mitosis at any given time. F r o m mid-proembryo stages through differentiation, 15.5 per cent of the total cells present are in division (Table 1). T h e percentage increases slightly to 17.4 per cent in embryos undergoing early maturation. W h e n embryos in stage 6 reach a length of more than 3 mm, however, the frequency of dividing cells drops to 5"5 per cent, and presumably goes even lower, later on, as the embryos enter dormancy. Cell size decreases during p r o e m b r y o development, especially in the course of the first few divisions. As a result, embryo length and embryo volume increase slowly at first, n o t reaching double the length or volume of the zygote until approximately the 32- and 16celled stages, respectively. T h e n both increase at a more rapid rate (Fig. 2). T h e changing, irregular shape of the embryo during differ-

272

L.W. MERICLE and R. P. MERICLE

•

Controls f o r embryos I r r a d i a t e d as early or mtd~proembryoa

•

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I 144

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103 168

T~e m hrs.

a

b

c

d

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1

8 r a g e in embryogeny

FxG. 2. Increases in m e a n e m b r y o length, cell

number, embryovolume,a n d

total n u c l e a r

volume as related to embryonic age and stage of development during the proembryo period of normal barley embryogeny. Based upon size and cell number comparisons with stage and age in 64 non-irradiated, control proembryos (stages and early differentiating embryos (stage 1). The plotted points for embryolength andvolume are derived specifically from 31 embryos acting as controls for ones irradiated in early and mid-proembryo stages, and those for cell number, from 41 embryos grouped by multiples of embryo length.

a-g)

entiation makes calculation of embryo volume impractical beyond the ~2000-celled stage. Embryo length, however, continues to increase throughout differentiation (Fig. 3) even to the final stages of maturation.(3) Between the zygote and late proembryo stages, the mean nuclear volume per cell drops by 8.5-fold.(is) I f these nuclear volumes are used to estimate the total nuclear volume per embryo in the control material of this study, there appears to be little

if any increase through the 4-celled stage, with doubling of the zygote nuclear volume not achieved before the 8-celled stage is reached (Fig. 2). Throughout the remainder & t h e proembryo period, the calculated total nuclear volume per embryo increases more rapidly (Fig. 2) but still at a somewhat lesser pace than cell number. Although total nuclear volume has not been estimated for differentiating embryos, it would be expected to parallel the increase in

CYTOLOGICAL

CONSEQUENCES

OF PROEMBRYO

IRRADIATION

273

Table 1. Proportion of ceUs in division in barley embryosfollowing acute X-irradiation (450 R) during proembtyo development and in non-irradiated control embryos

Irradiated as proembryos

Non-irradiated as proembryos

Embryonic stages scored

No. embryos scored

Stages e---5 Stage 6, < 3 mm Stage 6, >__3mm Stages e--5 Stage 6, < 3 mm Stage 6, >__3mm

85 15 -56 11 9

2000r

No. cells scored

57,171 15,000 -35,491 11,000 9000

Prop. cells in division ~ 4- s.s. 0.156-4-0-002 0.084±0.002 0.155 10"002 0.1744-0.004 0"05510.002

. maturingembryos

1800

1800

1400

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120C

.

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400 100

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0

embryos

50

i00

150

200

!

*

,

250

300

350

Time, hour8 FIO. 3. Compar~on of embryo length with embryo age and stage of development during normal barley embryogeny. Based on measurements of 74 non-irradiated, control embryos.

274

L . W . M E R I C L E and R. P. M E R I C L E

Table 2. Comparison of subsequent embryo development foUowing acute X-irradiation (450 R) of early vs. mid-proembryo stages Days postradiation

Range of embryo stages

0

a--b a--b b--d b--d + d+--d-e c--e e--g f--g+ e-f--l-2

rad con

1 rad con 2

rad con

4

rad con

5

rad con

7

tad con

Mean embryo length

Mean cell number

Evidence of abortion

45V 40

1.6 1.4

64* 51

5.7 6.7

some

79 74

9.3 30.5

some

Range of embryo stages

Mean embryo length

Mean cell number

e--f e--f e--f-g

138~ 136

218 301

Evidence of abortion

177 249

229* 1025

none

228* 412

646

none

1--4

f--1 +

e-f--g

158 164

354 408

some

I--3 4---5

563* 795

none

245 337

1388 2340

none

1--4-5 4-5--6

709* 1164

none

g--2 f+--5 4--5

570 842

noffe

°

Values for non-irradiated, control embryos (con) of comparable age, same initial stages, and similar mean sizes and cell numbers are shown below values for irradiated embryos (rad). Based upon 38 irradiated (and surviving) early proembryos, 35 irradiated mid-proembryos, and groups of 40 and 33 control embryos, respectively. *Mean embryo size or mean cell number significantly different (P<0.05) from that of non-irradiated, control embryos.

Table 3. Changes in mitotic cycle time followbzg art acute X-radiation exposure of 450 R at specific stages of proembryo development in barley Embryonic stage irradiated

Hours postradiation

Irradiated embryos

Early proembryo (stages a-b)

0 - 26 26- 51 51- 91 91-123

1.6 5.7 9.3 354.0

Mid-proembryo (stages e-f)

0 - 24 24- 48

218.0 229.0

NO

,h!"

Non-irradiated embryos

Tm*

NO

5.7 9.3 354.0 1388.0

14.2 35.4 7.6 16.2

1.4 6.7 30.5 408.0

6.7 30.5 408.0 2340.0

11.5 11.5 10.7 12.7

229.0 646.0

337.9 16.0

301.0

1025.0

13.6

Based upon the same groups of embryos shown in Table 2. t(log 2) *Tm = log N - log No

where: Tm t No N

= = = =

mitotic cycle time in hours no. of hours between sampling times mean no. of cells in original sample mean number of cells in last sample.

,IV

Tm*

CYTOLOGICAL CONSEQUENCES OF PROEMBRYO IRRADIATION cell number because nuclear volume per cell plateaus at essentially late-proembryo values.Cls)

Embryogenyfollowing irradiation During the first day following acute irradiation, early proem&yos proceed through one or more cell divisions with a nearly normal mitotic cycle time and concurrently undergo considerable cell enlargement (Tables 2 and 3). By the 1st day post-radiation (dpr), the irradiated embryos are sigifificantly (P<0.05) larger than unirradiated, control embryos of the same age, ahhough comprised of almost the same number of cells (Table 2). Marked prolongation of the mitotic cycle ( ~ 3 times normal length) occurs between the 1st and 2nd dpr (Table 3).

As a result, the mean cell number in irradiated embryos at the 2nd dpr is less than one-third that of the controls, but embryo size is essentially normal because of persisting cell enlargement (Table 2). Many of the irradiated early proembryos undergo only a few cell divisions, never increase any further in size, die, and eventually degenerate. Aborting embryos are common in 1st through 4th dpr samples with the highest incidence seen at the 2nd dpr. A few of the embryos wkich survive beyond this point may continue to undergo some cell divisions, but their cell number and stage of development lag far behind that of the controls. Development terminates with death and degeneration, usually before the onset of differentiation. Such

I r r a d i a t e d as EARLY PROEHBRYOS

1400

1400

Line = mean size

Shaded a r e a = s i z e r a n g e

1200 ~

1200

i 1000

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800

600

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2

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96

120

Hours p o ~ t - r a d i a t l o n

= 144

275

' 168

0 0

24

48

72

96

120

Hours p o s t - r a d l a t l o e

Fro. 4. Growth in length of barley embryos following acute X-radiation exposure (450 R) at early or mid-proembryo stages of development as compared with that of non-irradiated controls. Based on 38 irradiated (and surviving) early proembryos, 35 i,'radiated midproembryos, and groups of 40 and 33 control embryos, respectively.

276

L. W. MERICLE and R. P. MERICLE

embryos represent the lowermost part of the embryo size range in Fig. 4. Most of the early proembryos which survive beyond the 2nd dpr do not die. Instead, they enter a period of rapid cell division (Table 3) and increase in size (Fig. 4), followed by a resumption of nearly normal mitotic cycle time during the 4th-5th dpr. These embryos may continue to lag somewhat behind the control embryos (in terms of size and cell number) throughout the remainder of the proembryo period and on into differentiation. Many, however, remain well within the lower limit of size and cell number variability for unirradiated embryos, and their developmental stages overlap considerably with those of the controls (Fig. 2 and Table 2). Grossly abnormal, tumorous embryos are only rarely observed in dissected materials of the 3rd through 15th dpr samples (Table 4). Acute irradiation of mid-proembryo stages, by contrast, results in almost no gain in mean cell number during the first day post-radiation (Tables 2 and 3). By the 1st dpr sample, the mean cell number in the irradiated embryos has become significantly reduced below that of the controls; because of concomitant cell enlargement, mean embryo size is not significantly different (Table 2). The irradiated embryos are actually larger than unirradiated embryos would be, were they, too, comprised of the same reduced cell number, and are not significantly smaller than control embryos of the same age having more advanced ontogeny and severalfold more ceils. As cell enlargement attenuates and mitotic cycle time returns to nearly normal during the 2nd dpr, the size of the irradiated embryos becomes significantly less than in the controls (Tables 2 and 3). Most of the irradiated embryos then resume essentially normal rates of growth, parallelling the unirradiated embryos but remaining significantly smaller and set back about one day in development. As early as the 2nd dpr, there is no overlap of developmental stages between the embryos in the irradiated and control groups, and none in size by the 5th dpr (Fig. 4 andTable 2). Grossly abnormal, tumorous embryos are often observed in dissected material from the 4th through the 15th dpr (Table 4). In embryos irradiated as either early or mid-

proembryos and sampled between mid-proembryo and later differentiation stages, the percent of cells in division is not significantly different from that of the controls (Table I). But in contrast to the controls, the irradiated embryos do not reach stage 6 until after the 9th dpr. By the 15th dpr the irradiated embryos are in stage 6 and the percent of dividing cells has dropped, significantly, to ~ 8 per cent (Table 1). This reduction in frequency is similar to the decrease to ,,~5 per cent seen in unirradiated embryos of the same age, although taking place in irradiated embryos which are still less than 3 mm in length.

Chromosome aberrations The frequencies of mitotic bridges, chromosome fragments, and micronuclei observed in squash preparations of embryos sampled at v a r i o u s times following acute irradiation of of early and mid-proembryo stages are shown in Table 4. Information is lacking for the 1st and 2nd dpr samples of irradiated early proembryos because in these samples the embryos had either aborted or were lost during dissection and processing due to their extremely small size. Moreover, sectioned material is generally unsuitable for obtaining the needed data on a quantitative basis. Irradiation of mid-proembryos increases the incidence of mitotic bridges, chromosome fragments, and micronuclei to levels above the 95 per cent confidence intervals (C.I.95) of the pooled control material, when such intervals are recalculated in terms of the smaller cell populations present in each of the post-radiation samples. The frequency of mitotic bridges among anaphase and early telophase cells increases in the 2nd dpr samples to more than double the control values, and maintains a varying level above the controls through the 9th dpr. Chromosome fragments are most common in the Ist dpr sample, but have disappeared by the 4th dpr despite the continuing high frequency of mitotic bridges. Micronuclei are the most sensitive cytological indicator of radiation damage in this material, increasing in frequency at the I st dpr, reaching a maximum of more than 200 times the control value in the 2nd dpr sample, and finally declining to control

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278

L. W. MERICLE and R. P. MERICLE

levels after the 9th dpr. Examination of sectioned preparations of differentiating embryos reveals that micronuclei are present in cells comprising the actively dividing shoot and root regions, as well as in the more quiescent coleoptile, coleorhiza, and scutellum. Embryos irradiated as early proembryos show a very different response pattern. Mitotic bridge frequency is increased above the control level only in the 9th dpr sample. The incidence of chromosome fragments is significantly higher only in the 3rd dpr sample (although more than double the control value on the 15th dpr as well). Micronuclei provide by far the most divergent results between early and midproembryo irradiations, since in none of the samples from irradiated early proembryos is the frequency of micronuclei higher than that of the controls! Furthermore, examination of sectioned preparations indicates that micro-_ nuclei are absent in ceils of the embryo proper on the 1st and 2nd dpr also. That this absence is not just an apparent one--because of their not being observable in sectioned preparations-is shown by the fact that micronuclei are readily apparent in ovary tissue surrounding the irradiated early proembryos at the 1st and 2nd dpr and can be seen (though not accurately quantitated) within cells of the embryo proper in sectioned preparations of mid-proembryos.

ment (particularly in regard to the shoot region, Table 5). R-1 sterility and recessive mutation rates in the R-2 generation are generally highest following irradiation of mid-proembryo stages. (,0) Yet, embryo abortion in the R-1 generation is rare, and non-germination and seeding death occurs with low frequency.O6,1s,20) Similar associations between high incidence of developmental abnormalities and low abortion rate have been observed with multi-cellular proembryos of Arabidopsis(21) and Nicotiana.~ 7) It could be reasoned that viability of the organism is retained because the large number of cells, together with their relative developmental indeterminism at this time in embryogeny, permits viable cells (perhaps even ones normally 'destined' to become a part of some other organ) to substitute for those cells most severely damaged or killed. Present indications are, however, that survival of irradiated mid-proembryos depends more often upon their ability to 'make do' with fewer ceils. The barley embryo's capacity to function with a reduced meristematic capital has been amply demonstrated: irradiation during embryogeny or at maturity can reduce the number of cells normally contributing to particular regions without precluding survival. (12,18--20) Chromosome aberrations with attendant loss of genetic material are often implicated as the major cause of slowing or cessation of growth in irradiated meristems (see discussion by EVANS DISCUSSION and SPARROW).(11) More recently, EVANS(1°) The results of the present experiments has suggested that blockage of cells entering indicate that irradiation of developing barley mitosis (followed by premature differentiation, embryos at early vs. mid-proembryo stages senescence or cell death) accounts for the produces cytological responses as contrasting as greatest amount of effect, with recoverable those which we have already reported for mitotic cycle delay and chromosomal aberragenetic, histological, and physiological criteria. tions per se probably playing less important roles (18,20) than those usually ascribed to them. In irradiIrradiated mid-procmbryos, like plant meri- ated mid-proembryos of barley, the persiststems, generally,(x°) can apparently tolerate ently smaller embryo size and the ,,~ 1-day considerablc genetic (including chromosomal) set-back in development during later embryodamage and still remain viable. Cytological geny (Fig. 3) can be largely attributed to that aberrations (especiallymicronuclci) occur with period immediately following irradiation when high frequencies (Table 4) ;signsof cell death arc little gain in cell number is realized (Table 2). often seen; anomalous organogencsis is com- The cell enlargement evident at the Ist dpr mon.(16,18) The embryos exhibit retarded suggests mitotic delay, but the embryos show no growth throughout the remainder of embryo- reduction in the proportion of dividing cells. geny (Fig. 4), and subsequent seedling develop- Hence, any alteration in mitotic index must

CYTOLOGICAL CONSEQUENCES OF PROEMBRYO IRRADIATION

279

Table 5. Seedling growth data summarized from a depth dose s°Co study (Meride and Meride, unpublished) in which early and mid-proembryos were given a single acute exposure 0f800 R e°Co gamma radiation at 19.5 R/min

Hours posthydration Coleoptilc height, mm Shoot height, mm

Root length max., mm

Root number

72 96 120 72 96 120 48 72 96 120 48 72 96 120

Proembryo stage irradiated Early % C* Mid ~q-s.E. ~S.E. 9.31 q- 1.20~ 19.63±1.89t:~ 22.08-4-1.90 t 7.37±I.13~ I9-27 ±2.38~f:~ 36.48±4"07t$ 16-83±1.24~f~ 33"29±2"051"~ 42.25-t-2"601" 49.674-3.191" 3.374-0.24I" 4.31+0"22I" 4"56±0"22t 4.69±0"22t

79 58 57 89 7I 64 73 64 55 53 61 72 73 73

0"55-4-0.21t~ I 1-40±1.471"~ 20.85-4-2.05I" 0"17-4-0.111"~ 7"62±1-18I"$ 22-09±2.88t: ~ 10.45 4-0-99t:~ 24.43± 1.68t:~ 40.53-{-2.74 t 58.15±4.02t 2.83-4-0.251" 4.154-0.24 I" 4.55 ±0.25"~ 4.74-t-0.26I"

~oC* 5 34 54 2 28 39 46 47 52 62 51 69 72 74

Non-irradiated Controls ~±s.~. 11.81±0.78 33.83-4-0.50 38.87±0.37 8.31±0.59 27-09±1.02 56.91±1.32 22.96+0.57 51.69±1.79 77-26±3.49 93.15±4.19 5.544-0.13 5.98±0.04 6.28±0.07 6.414-0.09

*Means for irradiated material expressed as percent of control mean. l"Proembryo values significantly different from those of the control at 5% level. ~Proembryo values of early vs. mid-proembryos significantly different from one another at 5% level. have already been corrected. In view of the signs of cell death, it is likely that an actual loss ofcells takes place, nearly equaIling the number produced. Embryo growth in size and cell number resumes between the 1st and 2nd dpr. Both mitotic cycle time and percent of cells in division are essentially normal--despite this being the period of highest cytological aberration frequency (Table 4). The presence of chromosome aberrations, per se, therefore, does not produce growth retardation. O n the other hand, the gross genetic deficiencies which they can engender may be responsible for cell loss immediately following irradiation and for the precocious drop in dividing cells late in embryogeny. None of the mechanisms enumerated by EVANSO0) seem appropriate to account for the reduced shoot length during subsequent seedling development. This reduction, due mostly to a delay in emergence rather than a slower rate of seedling growth, may be totally unrelated to mitotic events, or even have its origin extraembryonically. (i,i~) W c earlierpostulated that the high frequencies of anomalous organogenesis which occur follow-

ing irradiation of mid- and late- proembryo stages could be due to inter- or intra-tissue incompatibilities arising from the presence of relatively high levels of radiation-induced genetic heterogeneity.O8) Here, chromosomal aberrations undoubtedly can play a most important role. T h e y may be implicated also in the abnormal DNA composition which C~ano (e} found at embryonic maturity following irradiation of late proembryo stages. Similar alterations were not observed after irradiation of some other stages not so prone to the production of organ abnormalities. Our new cytological data from irradiated mid-proembryos show that products of chromosomal damage are present in high frequency just prior to the onset of embryo differentiation and continue to be observable during later differentiation stages, even within the very regions of the embryo actively engaged in root and shoot formation. Since organ anomalies are also a common manifestation of radiation damage in plant meristems, generally, their bases too may lie in the genetic heterogeneity brought about by radiation-induced chromosomal aberrations.

280

L. W. MERICLE and R. P. MERICLE

In diameteric opposition to the situation with mid-proembryos, all evidence points to the virtual impossibility of young proembryos surviving in the presence of gross chromosomal damage. Their high abortion rate(iS, 2°) coupled with near absence of cytological aberrations in cells of viable embryos (Table 4), suggests that most cells which become grossly deficient, genetically, quickly die and are eliminated. Those embryos which survive and are capable of entering differentiation must be comprised almost entirely of cells which originally escaped major damage or are embryos reconstituted from one or a few relatively undamaged, highly totipotent, remaining cells. Such strong diplontic selection does not characterize all of embryogeny. Witness, for example, the subsequent appearance of mitotic bridges and chromosome fragments in differentiating embryos (Table 4). Nor does it operate as strongly against chromosome aberrations which result in no net mitotic loss of genetic information (e.g., transloeations not yielding dicentrics, inversions, etc.) or against point mutations (probably including small deletions). The sterility exhibited at R-1 sporophytic maturity and the recessive mutation rates expressed in the R-2 generation(20) are, however, both generally lower in frequency than following mid-proembryo irradiation. To what extent these or other factors contribute to the high frequency of non-germination manifest by irradiated early proembryos(2,1s,2°) and their progressively slowed growth rates during subsequent seedling development (Table 5) is not known. One of the most striking features of radiation response by early proembryos of barley or other higher plants, is the nearly complete absence of severe organ anomalies among those embryos surviving into differentiation stages.OS) We earlier postulated(is) that this was due to the relatively high degree of genetic homogeneity which would ensue for any genetic change produced by irradiation of what amounts to a single-celled system. Stage a is, of course, the one-celled zygote, and of the two cells comprising stage b, only the terminal cell normally contributes to formation of the shoot and essentially all of the root region.(s) Even with mitotic segregation of changes induced at

chromatid levels, large areas of the embryo could still be expected to share the same altered genome and thus escape incompatibilities which might arise from a high degree of genetic heterogeneity. Evidence that relative homogeneity for induced genetic changes can and does occur is provided by the large isomutantcarrying sectors which are obtainable following irradiation of early proembryos.(~9) The lack of alteration in DNA composition at embryonic maturity, despite significant depression of total DNA content, suggests also an absence of severe metabolic disturbance.(6) More recent genetic data(20) indicate, however, that genetic homogeneity does not always ensue after early proembryo irradiation. Moreover, the present study shows products of chromosome aberration to be present during the differentiation period in embryogeny (Table 4). From this we conclude that neither gross chromosomal deficiency nor genetic heterogeneity per se causes differemiation abnormalities when present du~ng active organ formation. The key to anomalous organogenesis seems to lie, instead, in the virtual absence of heterogeneity for gross chromosomal loss in the irradiated early proembryos vs. its peak incidence in the irradiated mid-proembryos during those stages of embryogeny just prior to or at the very onset of organ differentiation. It is particularly significant that these stages coincide precisely with the 'critical periods' for specific organ abnormalities reported earlier(a6,18) which notably, also, did not extend into the times of actual organ formation. The expectation that irradiation of early proembryos would lead to non-chimerism of the mutant-carrying state among all precursor cells of the generative tissue was the basis for our original interest in proembryo irradiation for mutation breeding programs.O0) GAUL'S(13) recent advocation of zygote and pollen irradiation was based on the same premise. Mutantcarrying sectors encompassing the entire generative tissue of the mature sporophyte can be induced with relatively high frequency by irradiating early proembryos.(ls,~°) Their occurrence is also possible, though rare, following irradiation of mid-proembryos.(Xs, ~°) Very surprising though has been the repeated observation that early proembryo irradiation often produces

CYTOLOGICAL CONSEQUENCES O F PROEMBRYO IRRADIATION sectional chimerism within the generative tissue. In some cases the sector sizes are not very different from those produced in ~ 1000-celled late proembryos wherein 2-4 cells probably constitute the generative tissue capital. 120) These findings suggest that under normal circumstances the chromosomes of one-and two-celled proembryos are highly multi-partite, genetically, but become decreasingly so as proembryo development proceeds. If so, then many of the large isomutant sectors obtained by early proembryo irradiation may be instances of embryo reconstitution from one or a few surviving cells at what would otherwise be a multi-cellular stage of embryogeny. Had these cells already undergone some decline in partiteness, the result would be an increase in sector size to levels once thought representative of the normal situation. The high genetic multi-partiteness exhibited by early proembryos of barley could be a reflection of unusual DNA content and/or structural state of the chromosomes. Although sometimes reported for embryonic and meristematic cells of higher plants, DNA contents above 4C and radiobiological partiteness of the chromosomes above 2--4 have not been widely accepted. The present study gives additional support to the possibility that the first few divisions of the zygote entail mostly a partitioning of cytoplasmic and nuclear components with little synthesis of new material.OS) One new line of evidence is the ability of early proembryos to undergo at least a few mitoses following irradiation, even if they are soon to abort. A second, is the lack of any appreciable increase in total nuclear or cell volume per embryo until after the 4-celled stage and no doubling until the 8- and 16-celled stages, respectively. In early cotton embryogeny, total cell volume actually decreases.~ la) It may be that the early proembryo's genetic multi-partiteness is due to DNA levels several-fold higher than the usual 2C-4C-2C transitions characteristic of somatic ceils, generally. Jensen'sOa) inability to induce *Difficulties in achieving 3H-thymidine incorporation into the DNA of shoot apices has been interpreted somewhat differently by VAN'T Hov (~) as probably due to the presence of large pools of precursor substances.

281

tritiated thymidine incorporation by cotton zygotes certainly implies that early embryo development does not involve DNA synthesis.* Chang's report of high rates of s2p incorporation into DNA during early proembryo development in barley14) and its marked depression during the 1st dpr(5) seems contradictory to the above. However, his group of early proembryos initially encompassed stages a through c, whereas ours were comprised of stages a and b only. If active DNA synthesis recommences after several distributive divisions of the zygote, it could be this capability which Chang was measuring. On the other hand, the decline in nuclear volume per cell during proembryo development may result from partitioning or depletion of other nuclear components important to the spatial distribution of the DNA or structural state of the chromosome: their identity--whether histories, other proteins, or even water--is not known. We anticipate that our microspectrophotometric studies of barley proembryo development now underway will help to solve these issues and in doing so, shed further light on the causes of the radiation response differences between early and midproembryos. Acknowledgements The authors wish to thank SUSAN BEAUREGARD, RONALD BRANDON, SANDRA ENGLISH-and express their special appreciation to BERTA

NUNEz and SUSANVAN PEURSEM--fortheir valuable technical assistance during various phases of this study.

REFERENCES

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L. W. M E R I C L E and R. P. M E R I C L E

5. C~a~G C. W. (1967) Effects of ionizing radiation on nucleic acids during embryonic development --metabolism during embryogeny and at embryonic tissue level. Can. 07. Botany 46~ 51-56. 6. CHANGC. W. and MEPJCLE L. W. (1964) Effects of ionizing radiation on nucleic acids during embryonic development--L Quantitative and qualitative analysis at embryonic 'maturity'. Radiation Botany ~ 1-12. 7. DEwu~ux M. and SCAR~CIA MUGNOZZA G. T. (1964) Effects of gamma radiation of the gametes, zygote and proembryo in Nicotiana tabacum L. Radiation Botany 4, 373-386. 8. Etn~rusA. M. (1954) A study of the embryology of Hordeum vulgare L. and the embryonic abnormalities induced by X-rays. Ph.D. Thesis, Michigan State University, E. Lansing, Michigan. 9. EuNus A. M. (1955) The effects of X-rays on the embryonal growth and development of Hordeum vulgare L. 07. Exptl. Botany 6~ 409--421. 10. EVANS H. J. (1965) Effects of radiations on meristematic cells. Radiation Botany 5, 171-182. 11. EVANSH . J . and SPARXOWA. H. (1961) Nuclear factors affecting radiosensitivity. II. Dependence on nuclear and chromosome structure and organization. Brookhaven Symp. Biol. 14, 100-127. 12. GAUL H. (1961) Studies on diplontic selection after X-radiation of barley seeds. Effects of Ionizing Radiations on Seeds. Proe. Symp. Karlsruhe (IAEA & FAO, Vienna) 1960, pp. 117-138. 13. GAUL H. (1964) Mutations in plant breeding. Radiation Botany 4, 155-232.

14. JENSEN W. A. (1964) Cell development during plant embryogencsis. Brookhaven Symp. Biol. 16, 179-202. 15. MELETTIP., FLoras C. and D'AMATOF. D. (1964) Occurrence of an inhibitor in wheat endosperm as revealed by embryo transplantation in irradiated seeds. Radiation Botany 4, 497-502. 16. MERICLE L. W. and MERICLE R. P. (1957) Irradiation of developing plant embryos. I. Effects of external irradiation (X-rays) on barley embryogeny, germination, and subsequent seedling development. Am. 3. Botany 44, 747-756. 17. MEmCLE L. W. and M.~mCLE R. P. (1959) Changes in amino acid content induced by the X-radiation of developing barley embryos. Genetics 44, 526. 18. M~RICLE L. W. and MEmCLE R. P. (1961) Radiosensitivity of the developing plant embryo. Brookhaven Symp. Biol. 14, 262-286. 19. M.~mCLE L. W. and MEP~CLE R. P. (1962) Mutation induction by proembryo irradiation. Radiation Botany 1, 195-202. 20. MEmCLE L. W. and MEmCLE R. P. ~1967) Mutation induction as influenced by developmental stage and age. In H. Stubbe (ed.), Erwin Baur Memorial Lectures IV, 1966. Abhandl. Deut. Akad. Wiss. Berlin, pp. 65-77. 21. REINHOLZ E. (1959) Beeinflussung der Morphogenese embryonaler Organe durch ionisierende Strahlungen. Strahlentherapie 109, 537-554. 22. VAN'T HOFJ. (1968) Personal communication.