Relation of embryo structure, node position, tillering and depth of planting to the effects of X-rays in barley

Radiation Botany, 1962, Vol. 2, pp. 89 to 108. Pergamon Press Ltd. Printed in Great Britain.

RELATION OF EMBRYO STRUCTURE, NODE POSITION, TILLERING AND DEPTH OF PLANTING TO THE EFFECTS OF X-RAYS IN BARLEY* PATRICIA SARVELLA, R. A. N I L A N a n d C. F. K O N Z A K

Departments of Agronomy, Mississippi State University and Washington State University (Received 29 November 1961) Abstract--Biological, especially genetic, effects of X-rays in barley are influenced by embryo development, node position in MI plants, depth of planting and plant tillering. Bud or tillerinitial viability was determined with the vital stain, tetrazolium chloride. Both deeply planted and irradiated seeds exhibited reduced tillering from the bud in the axil of the coleoptile. Mutation rates (frequencies of chlorophyll-deficient Mz seedlings) were calculated by the M 1 plant, M1 spike, or M2 seedling methods. Mutation rates were also determined by each method for apical spikes, primary and secondary axillary spikes, and each node position. These latter rates differed less from one another when scored by the M~ spike or M2 seedling methods than when scored by the M 1 plant method. The M~ spike mutation curve approached closest to linearity for plants with all primary axillary and apical spikes present and with all duplicate mutants excluded. Reliable mutation rates can be calculated from the apical spike alone. Node position on the M 1 plants, with the exception of the fourth node, did not appear to affect mutation rates. Spikes above the fourth node and from secondary axillary tillers frequently had as many or more mutations than the primary axillary spikes. In fact, a higher mutation rate occurred in plants with higher numbers of tillers. Summarized mutation rates of the primaries at the upper nodes were higher than at the lower nodes. The converse was true for the secondaries which also had lower rates than the primaries. Fluctuations in rates were related to variations in embryo structure and to a decrease in the number of nodes (with tillers), spikes, and seedlings per plant. Therefore, the nearly linear mutation rates with increasing dose are probably related to high numbers of tillers which would permit mutant cells to be detected. R ~ s u m ~ Les effets biologiques sp6cialement g6nftiques des rayons X chez l'Orge sont influenc6s par le d6veloppement de l'embryon, la position des noeuds chez les plantes M l, la profondeur ~t laquelle on plante et le tallage des plantes. La viabilit6 de l'initiale du bourgeon ou du talle a 6t6 d6termin6e au moyen du colorant vital, chlorure de tetrazolium. Les graines plant6es profond6ment et les graines irradi6es ont tomes deux montr6 un tallage r6duit partir du bourgeon, dans l'axe du col6optile. Les taux de mutations (fr6quences de plantules M2 d6ficientes en chlorophylle) ont 6t6 calcul6s par la m6thode des plantes M1, des 6pis M1 ou des plantules M2. Les taux de mutations ont aussi 6t6 ddterminds par chacune de ces m6thodes pour les 6pis apicaux et pour les 6pis axillaires primaires ou secondaires et darts chaque position des noeuds. Ces derniers taux

*Scientific paper No. 2162, Washington Agricultural Experiment Stations, Pullman, Washington. Project 1002 and 1068. Work was begun at Washington State University with support from a postdoctoral fellowship and funds provided for medical and biological research by State of Washington Initiative Measure 171. Work was completed with funds made available by the Mississippi State Agricultural Experiment Station. 89

90

R E L A T I O N OF EMBRYO STRUC'I~URE T O T H E EFFECTS OF X-RAYS different moins l'un de l'autre lorsqu'ils sont recensds par la m6thode des plantes M,. La courbe des mutations des 6pis M1 est tr6s proche de la lin6arit6 pour des plantes dont tousles dpis axillaires primaires ou apicaux sont prdsents et lorsque l'on exclut tous les mutant dupliqu6s. Des taux de mutations d'une grande sflret6 peuvent 6tre calcul6s ~t partir de l'6pis apical considfr6 seul. La position des noeuds/t l'exception du quatri~me sur les plantes M1, ne semble pas affecter les taux de mutations. Des 6pis situds au-dessus du quatri~me noeud et des dpis provenant de talles axillaires secondaires avaient fr6quemment autant ou plus de mutations que les 6pis axillaires primaires. En fait, un taux plus dlev6 de mutations est apparu chez des plantes poss6dant des nombres plus dlevfs de talles. Les taux globaux de mutations des dpis primaires venant de noeuds supdrieurs 6taient plus 61evds que pour des noeuds inf6rieurs. Le rapport 6tait exact pour les secondaires qui ont aussi montr6 des taux moins 61evds que les primaires. Des fluctuations de taux sont rapportdes aux variations de structure de l'embryon et ~t une diminution du nombre de noeuds (avec talle), des 6pis et des plantules par plante. D6s lors, la relation presque lindaire entre les taux de mutations et les doses est rapportde aux nombres 61evds de talles qui permettraient la ddtection de cellules mutantes. Z u z ~ e n f a s s l u t n g - - B i o l o g i s c h e , besonders genetische Wirkungen der R6ntgenstrahlen auf die Gerste werden dutch Embryoentwicklung, Knotenposition in M~ Pflanzen, Einsaattiefe und Seitenhalmbildung beinflusst. Knospen- oder Seiten-halmeninitialenlebensf/ihigkeitwurde mit Tetrazoliumchlorid Vitalf/irbung festgestellt. Sowohl tief ges/ite wie auch bestrahtte Samen zeigten verminderte Seitenhalmbildungvonder in der Achsel der Koleoptile gelegeneu Knospe. Mutationsraten (Anzahl der Chlorophylllosen M2 Keimlinge) wurden mit den M1 Pflanzen, M 1 Ahren, oder M2 Keimlingmethoden berechnet. Mutationsraten wurden auch mit jeder Methode ffir die Haupt/ihre, prim/ire und sekund/ire Seitenhalm/ihren und jede Knotenposition berechnet. Die letzteren Raten unterscheiden sich von einander weniger wo mit der M 1 Ahren oder Ms Keimlingmethode berechnet als wir mit der M~ Pflanzenmethode. Die M1 ~hrenmutationskurve war ann/ihernd linear fiir Pftanzen die alle prim/ire Seiten- und Haupt/ihren hatten und mit allen duplikanten Mutanten ausgeschlossen. Zuverl~issige Mutationsraten k6nnen v o n d e r Haupt/ihre allein erechnet werden. Knotenposition der M1 Pflanzen, mit Ausnahme des vierten Knoten, scheint Mutationsraten nicht zu beeinflussen. )khren fiber den vierten Knoten und von sekund/iren Seitenhalmen batten oft so viele oder mehr Mutationen als die prim~ren Seitenhalme. In der Tat eine gr6ssere Mutationsrate erscheint in Pflanzen mit gr6sserer Anzahl yon Seitenhalmen. Zusammengefasste Mutationsraten der Prim/itCh der oberen Knoten waren gr6sser als die der Niedrigeren. Das Gegentibergesetzte geschah in Sekund/iren die auch niedrigere Raten als die Prim/iren halten. Ratenschwankungen waren mit Variation der Embryonenstruktur und Verminderung der Anzatfl der Knoten (mit Sprossen), Ahren und Keimlinge pro Pflanze verbunden. Deshalb sind die mit zunehmenden Dosis ann/ihernd lineare Mutationsraten wahrscheinlich mit der grossen Anzahl den Seitensprossen die die Entdeckung mutanter Zellen erlauben wfirden, verbunden.

INTRODUCTION IN RECENT years there have been m a n y studies o n r a d i a t i o n - i n d u c e d m u t a t i o n s in the barley seed. I n these studies, a reliable m e a s u r e of true m u t a t i o n s rates has been difficult to o b t a i n because of the lack of i n f o r m a t i o n o n c e r t a i n factors that influence passage of i n d u c e d m u t a t i o n s from the m u t a g e n - t r e a t e d seed to its progeny. After i r r a d i a t i o n the seed contains n o n -

m u t a t e d a n d m u t a t e d ceils. This chimeric n a t u r e is of p a r t i c u l a r i m p o r t a n c e i n tillerinitial cells since it is the s u b s e q u e n t generations of these cells that carry the m u t a t i o n s to the next p l a n t g e n e r a t i o n where they c a n be detected. GAUL(s) has recognized this chimeric p r o b l e m a n d its relation to tiller n u m b e r a n d development, a n d he has a t t e m p t e d to devise more accurate methods of scoring i n d u c e d m u t a t i o n s i n barley by taking into a c c o u n t

PATRICIA SARVELLA, R. A. NILAN and C. F. KONZAK influences of tillering and competition between mutated and non-mutated cells (diplontic selection). Previous studies with barley have also been hindered by the lack of detailed analyses of the relationship between the origin and ontogeny of tillers in irradiated seeds and the frequency of induced mutations. TbSs study was designed, therefore, to determine: (1) the effects of different depths of seed planting on the origin and development of tillers; (2) mutation and segregation rates in apical and primary and secondary axillary spikes; and, (3) if the apical spike alone can be used to measure mutation rates.

91

weeks in the greenhouse, the cartons were moved to the field, randomized, and sunk into trenches. After four weeks the plants were removed, fixed in FAA (eighteen parts ethyl alcohol, one part glacial acetic acid, one part formalin), dissected, and the number, location, and size of tillers recorded. A similar procedure was followed for determining the formation of tillers in irradiated seeds, except that only the one-inch planting was employed.

Mutation Experiment

In the spring of 1959, the seeds for mutation studies were divided into four replicates and irradiated. They were then germinated on blotters in petri dishes for two days and transMATERIALS AND METHODS planted into the field in fifty foot rows, spaced Resting barley seeds (Hordeum vulgate vat. 189 ft apart. Each seed was carefully planted Hulless Haisa) were used in this study. The). four inches apart since spacing is known to were genetically homogeneous as they were affect the amount of tillering(2) and at about a selected from the multiplied seed of a single one-inch depth. For each radiation dose in a plant. The seeds were stored for about two replicate there were two rows containing 125 weeks in a desiccator containing CaC12 to seeds. Treatments within replicates were ranreduce the moisture content to about 8 per cent domized. at the time of irradiation. At the end of two and one-half weeks The seeds were irradiated embryo side up in emergence frequency was scored as the number a single layer on a turntable to allow equal of plants above ground compared with the exposure to the X-rays. The turntable was number of seeds planted. Survival data were centered 42 cm below a beryllium window recorded as the number of plants at harvest A.E.G. Machlett X-ray tube. The tube was time compared with the number of seeds operated at 34 kV and 25 mA without filter planted. and the delivery rate was 1000 r/rain. In Viability of apical and axillary buds in some addition to the control, three exposures were seedlings from each dose of irradiation was given: 2,500 r, 10,000 r, and 15,000 r. Immedi- determined by the vital stain, tetrazolium ately after irradiation the seeds were placed in chloride. This chemical stained live buds red distilled oxygen-free water to minimize the while dead tissue remained white.07) The most after effects of irradiation. effective stain was a 0" 1 per cent solution of 2-(p-Iodophenyl) -3-(p-Nitrophenyl) -5-phenyl Depth of Planting Experiment tetrazolium chloride (I)r For sixteen days T o determine the influence of depth of seed before staining, the seedlings were grown in sowing on tillering, non-irradiated seeds were Hoagland's nutrient solution in petri dishes planted at one-, three-, and four-inch depths in under continuous fluorescent lights. Immedithe greenhouse. All M 1 plantings were done at ately before staining, the leaves covering the Washington State University in the summer of axillary buds and the apical meristems were 1959. These plantings were in half-gaUon milk removed. After this, the seedlings were stained cartons to facilitate removal of the plants for at 40~ for at least 15 min and the color of the examination. Half the seeds were planted buds observed. immediately, and the remainder were germiT o compare the actual amount of tillering nated for two days on blotters in petri dishes and with that indicated by the IdVT study, some then transplanted into the cartons. After two control plants and plants from 10,000 r treated

92

RELATION OF EMBRYO STRUCTURE TO THE EFFECTS OF X-RAYS

seeds were removed from the field after five weeks of growth. These plants were dissected and studied similarly to those in the depth of planting experiment. For the cytogenetic analyses, the origin of the tillers had to be determined in relation to the embryo anatomy. The mature embryo of the barley seed at the time of irradiation contains the apical and three axillary buds or meristemsO, 8.11) (Fig. 1). The apical bud gives rise to the apical spike (ap.) and the three axillary buds produce the three primary axillary spikes (pr. ax.) (Fig. 2). These axillary spikes correspond to the primary axiUary tillers from the first three nodes of the plant. Additional primary axillary buds are laid down after germination by apical meristematic activity at each node. All spikes derived from the same node are designated as a tiller group (t. g.) and are composed of the primary and one or more secondary axillary spikes. T h e main criteria that were used to (mAp.

/' 4

7'

2

SEC~'~ PR. AX.

FIo. 2. Schematic diagram of mature barley plant showing the tillering. Apical tiller and spike (ap), Primary axillal~/ tillers and spike (pr. ax.) and Secondary axillary tillers and spikes (see.). The numbers indicate the tiller groups (nodes) starting from the ground.

distinguish origin of the spikes were location of the scale leaves, orientation of the tillers to the apical spike, and alternation of the tiller groups on the apical spike. Tiller origin was determined after three weeks and rechecked at the end of another four weeks. After determining their origin, the apical spike and primary axillary tillers were marked with different colored tags. Tiller groups were numbered according to their origin in the following manner: spikes originating from buds in the axils of the coleoptile at the first rtode belonged to tiller group one, in the axils of the first foliage leaf or second node to tiller group two, etc., until all nodes producing tillers and spikes were numbered. When the spikes were harvested, the apical spike, the primary axillary spikes, and the tiller groups from each plant were stored separately. Chromosome aberrations in the microsporocytes were scored in spikes derived from the apical buds, and collected from control and 10,000 r treated plants. Aberrations were examined in acetocarmine squashes of microsporocytes and the mean quadrivalent frequency was calculated. A ring-of-four (IV) was given a value of one; a ring-of-six (VI), 1.5 and a ring-of-eight ( V I I I ) , two. I f the apical spike (or the first axillary spike, if the apical spike could not be studied in a plant) showed an aberration then as m a n y spikes as possible were collected from this plant. Frequencies of aberrations were calculated in relation to the n u m b e r of spikes examined per plant. For mutation analyses, M S seedlings were grown in greenhouses at Mississippi State University during the winter of 1959-60. Ten days after planting, the frequency of chlorophylldeficient seedlings was recorded for each M 1 plant according to GUSTAFSSON'Sclassification. (x~ In every treatment the M 1 spikes were counted and the total number of Ms seedlings was estimated by counting all the seedlings from every tenth M 1 plant (count plants) and the averages determined. Occasional bifurcated spikes were treated as one spike. Mutation rates were determined, first, for the total plant population by using the M 1 plant, M 1 spike, and M S seedling methods. Second, rates were calculated in several ways for complete plants (having aU apical and p r i m a r y

Fro. 1. Barley embryo in the resting seed. Note the three axillary meristems already formed in addition to the apical meristem. A. Photomicrograph of barley embryo (Slide lent by Dr. L. W. Mericle). B. Drawing of the apical meristem (Taken from LERMER and HOLZNER. (11))

FIG. l b

PATRICIA SARVELLA, R. A. NILAN and C. F. KONZAK axillary spikes present). In these plants, mutation rates were obtained for the total plant ("all spikes"), apical spikes, and primary and secondary axillary spikes within a tiller group. The mutation rates for "all spikes" of a complete plant were calculated by three methods: (a) counting all mutants even if there was more than one in the same plant; (b) scoring duplicate mutants only once within the four tiller groups corresponding to the four meristems present in the seed at irradiation, and (c) scoring all mutants per plant only once (duplicates were eliminated). I n the 10,000 r and 15,000 r treatments these ratios were calculated separately for each tiller group in a plant. Segregation frequencies of mutants from m u t a n t spikes were calculated separately according to origin of the spikes, i.e. for "all spikes", apical spikes, primary axiUary spikes, or all spikes of an entire tiller group. Statistical comparisons were made by an analysis of variance with arc sin transformations. Tests of difference between means were analyzed according to DAVIES' method(5) and other comparisons were analyzed by Chi-square. All differences between treatments that are stated in the results are significant unless otherwise indicated. EXPERIMENTAL RESULTS

Emergence and survival About three-fourths of the control seeds emerged (Table 1). Compared with the control, the frequency of seeds that emerged in the 2,500 r treatment was increased in the 10,000 r treatment, it was nearly the same; and in the 15,000 r treatment there was a sharp reduction. Survival after emergence was usually g o o d - usually only 2-7 per cent of the plants failed to mature. Table 1. Effect of radiation on emergence and survival to maturity of barley plants (average of four replicates) Treatment Control 2,500 r 10,000 r 15,000 r

~o Emerged* 75"37 82"67 74"77 59"81

% Survived* 72"94 80"76 71"83 52"47

*Based on the number of seeds planted.

93

Origin and number of tillers as influenced by sowing

depths Preliminary experiments showed a difference in tillering between seeds planted at different depths. Seeds planted at depths of one, three, and four inches were selected for study as a definite reduction in tillering occurred between the three- and four-inch plantings. Tillers in deeper plantings usually came from the second to fifth leaf node while those in shallower plantings (89 in. to 2 in.) came from the first to the fourth nodes. Tillers arose from as m a n y as six nodes. In the final experiment (Table 2), seeds planted at greater depths showed a considerable reduction in germination and emergence. This was especially true for seeds which were transplanted from petri dishes to milk cartons. T h e seeds in three- and four-inch plantings reacted in various ways: some were not found or had decayed; some did not emerge and grew parallel to the surface or else were squashed, coiled, or accordion-shaped. Root systems in the seedlings which had started to grow appeared normal. Plants from seed sown directly at three- and four-inch depths (Table 2) showed a reduction of tillering from the coleoptile bud compared with plants from seeds sown at a depth of one inch. As in the preliminary study, the fourth leaf bud exhibited some increase of tillering in plants from deeper-planted seeds. Transplanted seedlings from seeds that were germinated for two days in petri dishes exhibited a low survival rate. None of the eighty seedlings planted 3 in. deep survived and only a few (Table 2) survived planting at 4 in. These few survivors had fewer tillers than did plants from seedlings planted 1 in. deep. At the one-inch depth, plants from seeds planted directly into the ground produced more tillers from the bud in the axil of the coleoptile than did the transplants. However, the transplants showed an increase of tillering from the buds in the axils of the other leaves. At a depth of 4 in., plants from directly planted seeds had tiller frequencies at the different nodes similar to the transplants, although tillering was less in the transplants. Location and n u m b e r of tillers of plants from irradiated seeds were also compared with

94

RELATION OF EMBRYO STRUCTURE TO THE EFFECTS OF X-RAYS

Table 2. Influence of the various treatments on the location and number of tillersformed at different nodes depending on the depth of planting (average of two replicates) No. of plants

~ of plants with tillers in the axil of the

planted scored

Coleoptile 1st leaf 2nd leaf 3rd leaf 4th leaf 5th leaf

Treatment

Seeds planted directly into ground 1 in. deep 3 in. deep 4 in. deep

%

%

%

%

%

60 70 80

52 26 13

61.5 3.8 23.0

94.2 76.9 100-0

88.5 84-6 100.0

84.6 76.9 84.6

38.5 57.7 61"5

Seeds transplantedfrom petri dishes after two days 1 in. deep 4 in. deep

60 100

57 7

49.1 28-6

100.0 85.7

100-0 85.7

86.0 57.1

50"9 14.3

Seeds irradiatedplanted 1 in. deep 2,500 r 10,000 r 15,000 r

70 90 200

60 74 151

53.3 20.3 17"2

95.0 93.2 87.4

91.7 95-9 82.1

75.0 77.0 62"2

25"0 17"6 23"8

49 49

63.3 46.9

100.0 98.0

100.0 98-0

100-0 98-0

87"8 67.3

Field plants dug up Control 10,000 r

results of the above two experiments. Plants from seeds treated with 2,500 r and planted 1 in. deep produced a tillering pattern similar to plants from non-irradiated seeds planted at the same depth. A dose of 10,000 r, however, reduced tillering from buds in the axils of the coleoptile and the fourth leaf. A dose of 15,000 r further reduced tillering from the coleoptile bud and markedly reduced tillering at the third leaf bud. At this dosage, other leaf buds also showed a slight reduction in tillering compared to leaf buds treated with lower dosages. At the end of six weeks, the extent of tillering in seedlings transplanted directly into the field from petri dishes was compared with that in seedlings raised in milk cartons (Table 2). The fieldgrown control plants and plants grown in milk cartons showed similar characteristics for the one-inch deep planting. A higher per cent of buds had formed tillers at the coleoptile and fourth leaf nodes. With the 10,000 r treatment, however, plants grown in the field tillered better than plants grown in milk cartons, although

%

34.7 26.5

these field-grown plants did not tiller as well as the field-grown controls. Furthermore, the fourth leaf bud in the irradiated field plants showed a marked reduction, and the coleoptile and fifth leaf buds showed some reduction of tillering compared with the controls. Tetrazolium chloride studies The application of tetrazolium chloride ( I N T ) to dissected primordia of non-irradiated seedlings affected buds according to their location. The per cent of stained buds (Table 3) was lowest at the coleoptile node, increased at the first leaf, and was highest at the second leaf and the apical meristem. Twin buds occasionally were observed in the axils of the coleoptile or the first leaf. As the buds received increasing X - r a y dosages, the per cent of stained buds either was reduced or remained almost the same. One exception was a prominent increase of stained buds in the axils of the second leaf in the 2,500 r treatment. There was a marked reduction of

PATRICIA SARVELLA, R. A. NILAN and C. F. KONZAK

95

Table 3. Frequencyof buds at different nodes stained by tetrazolium chloride Buds stained red (viable) No. of plants % staining red control 2,500 r 10,000 r 15,000 r

180 190 185 191

in the axil of the

apical meristem

Coleoptile

1st leaf

2nd leaf

% 11"1 I0"0 11"9 13"1

% 33"3 32"6 30"3 23"0

% 92"8 97.4 78-4 54-0

stained buds at the second leaf node in seedlings from both the 10,000 r and 15,000 r treated seeds compared with seedlings from the control and 2,500 r treated seeds. In the 15,000 r treatment, the per cent of stained buds was reduced at the first leaf node as well. The per cent of stained apical meristem buds varied the least. Field-grown plants and plants from the irradiated seeds grown in milk cartons exhibited a higher per cent of tillering than would be expected from the per cent of stained buds in the axils of the coleoptile and first Ieaf (Tables 2 and 3). T h e buds at the second leaf node showed more damage from irradiation in the tetrazolium chloride study than in the tillering study, but a higher per cent of the apical meristems were stained compared with the amount of tillering at the third leaf node. Twin tillers which probably arose from twin buds seen in the I N T study were observed occasionally in the axils of the coleoptile and first leaf. Chromosome interchanges The per cent of interchanges in apical spikes of M 1 plants was increased by a dose of I0,000 r (Table 4). The per cent of apicals containing "rings-of-four" in the first two replicates was 12.2. Plants from the 10,000 r group known to contain interchanges in either an apical spike or the first analyzable axillary spike were further analyzed for interchanges in axillary spikes. The 20 per cent interchange frequency for these axillary spikes was higher than the frequency in the apical spikes. Plants with known interchanges were divided into three groups depending on the number of

% 90"0 86"3 88"6 83.8

spikes studied per plant (Table 5). If the apical spikes were included, the per cent of spikes with interchanges decreased as the number of spikes per plant increased. The decrease was significant for the five to eight spike group and highly significant for the nine to sixteen spike group. It would, therefore, appear that since interchange frequency decreased, the mutant cells were being overgrown by the normal cells (diplontic selection) during development of the tillers and spikes. However, if one spike corresponding to the apical spike and known to have an interchange (Table 5) was removed, then the frequencies of interchanges between the three groups showed no significant difference. Total frequencies of interchanges in the two groups with the most tillers and spikes were similar to the frequency in apical spikes of the general population (Table 4). Analyses of mutation rates Since frequency of tillering was considered to be an important factor in determining and calculating chlorophyll-deficient seedling mutation rates, plants were chosen which had the apical spikes and all primary tillers with spikes present (complete plants). Numbers of total plants and complete plants and the average number of spikes per plant for the different treatments are presented in Table 6. The number of spikes was only slightly higher for complete plants than for all plants within a radiation treatment. Compared with the control treatment, the 2,500 r and 10,000 r of X-rays slightly decreased the spike n u m b e r while the 15,000 r treatment markedly decreased

96

RELATION OF EMBRYO STRUCTURE TO THE EFFECTS OF X-RAYS Table 4. Comparison of the location of the spikes on the XI plants to the per cent of chromosomal interchanges*

Plants with known interchanges

All plants No. of plants studied

~ Apicals with interchanges

Av. of 4 reps. control 10,000 r

249 270

2"0 13"0

Av. of 2 reps. 10,000 r

188

12"2

No. plants

Av. no. of spikes with interchanges per plant

Total ~ spikes with interchanges

% Axillary spikes with interchanges

46

1.9

26"2

20"6

*Calculated on a "ring-of-four" basis, i.e. a ring-of-6 was considered as 1"5 and a ring-of-8 as 2.

Table 5. Chromosomal interchanges in spikes or M1 plants with known interchanges. (10,000 r.)

Apical spikes* included No. of spikes per plant studied 1-4 5-8 9-16

No. of spikes

No. spikes with interchanges

34 182 95

16 48 18

Apical spikes* excluded No. of spikes 24 154 87

No. spikes with interchanges 6 20 10

*Interchange may have been determined in an axillary undergoing meiosis.

it. Average number of tiller groups in the complete plants and number of seedlings per spike in the count plants also decreased as the dose increased. Spikes from control plants had about twice as many seedlings as the spikes in the 15,000 r treatment. There was no significant difference between the number of seedlings from spikes with mutants and the number of seedlings from the total spikes in the count plants. Mutation rates by M 1 plant method Mutation rates in seeds irradiated at the three X-ray dosages were calculated by the M 1 plant method for complete plants (Fig. 3). These rates, depicted as curves, are for spikes from apical spikes, primary and secondary axillary tillers, the first four tiller groups, and the total

population. The mutation rates decreased with X-ray dose as more mutants were excluded because of grouping. From 10,000 r to 15,000 r, all curves except for spikes from secondary axillary tillers (Curve C) increased slightly. Curves of the mutation rates for apical and primary axillary tillers approached linearity with dose. Apical and primary axillary spikes thus appear to be fairly good indicators of the mutation rate by the M 1 plant method. The primary axillary mutation curve (B) was calculated from mutations in spikes derived from four different meristems in the seed at irradiation--the three axillary meristems in different stages of development and the apical meristem which give rise to subsequent primary axillary meristems. Depending on the number of tiller groups, the mutation rate in the

PATRICIA SARVELLA, R. A. NILAN and C. F. KONZAK

97

Table 6. Number of tillers in all plants and complete plants with all apical and primary axillary spikes present. (Average of.four replicates) All plants

Chosen plants

No. of population plants

Av. no. spikes per population plant

No. of complete plants

723 803 731 530

17.55 16-89 15"92 10.64

595 716 574 331

Control 2,500 r 10,000 r 15,000 r

"9

Z

~

ne lal Q.

ua ne

~'

OZ "3

."

/ s

S'.f"

o

2,300

I

~, ~'SS

~o,boo

m,6oor

Fro. 3. Frequency of M2 seedling mutants in complete plants at 2,500 r, 10,000 r, and 15,000 r calculated by the M1 plant method (av. of 4 reps.). A. Apical spikes, B. Primary axillary spikes, C. Secondary axillary spikes, D. Spikes of first four tiller groups and E. "All spikes", every mutant counted. primary axillary spikes should, therefore, be at least four times greater than the rate of the apical spikes. Rates for the 10,000r and 15,000 r treatments, however, increased three times the apical rate, although the 2,500 r dose caused a four-fold increase. An analysis of variance comparing mutation rates for apical and primary axillary spikes

Av. no. of tiller groups Av. no. Av. no. (nodes)per of spikes seedlings complete per complete per spike in plant plant count plants 5-35 4-67 4.26 3.61

17.81 16.97 16.50 11.70

24-64 21.14 19"95 13.19

Av. no. seedlings in mutant spikes 23.25 22.35 15.10 13.09

showed a hJgh/y significant difference between X-ray doses, the two methods of computation (apical vs. primary axiUary), and their interaction. The C.V. was 24.9 per cent. There were no significant differences in mutation rates between apical spikes of the control and 2,500 r treatments, and between apical spikes of the 10,000 r and 15,000 r treatments; but between these two groups there was a highly significant difference. Mutation rates in the primary axillary spikes showed a difference at the 1 per cent level between the control, 2,500r, and 10,000 r values. It can be seen from Fig. 3 that the rates did not vary greatly for spikes from the secondary tillers (Curve C). Mutation rates were also calculated by the M 1 plant method for "all spikes". When three techniques of computation--aU mutants, Curve E; all duplicate mutants excluded; and tillergroup mutants, Curve D (Fig. 3)~-were compared, a highly significant difference was found between dosages. However, between the 10,000 r and 15,000 r treatments, the rates did not differ. Differences in mutation rates between the three techniques of computation were also highly significant. Yet, the two analyses, the tiller group mutants, and exclusion of all duplicate mutants, did not differ in mutation rates. Mutation rates obtained by counting all mutants differed at the 5 per cent level from rates obtained by the other two techniques of computation. The C.V. was 13-3 per cent. The interaction of techniques of computation x doses was not significant which indicates that the three

98

RELATION OF EMBRYO STRUCTURE TO THE EFFECTS OF X-RAYS

techniques for scoring mutations produced similar results from one dose level to another. Mutations per tiller group Mutation rates also were calculated by the M I plant method for the n u m b e r of tiller groups in a plant using only tiller groups in the complete plants which were represented by more than twenty-five plants. Mutation rates for each tiller group at the three X-ray dose levels are plotted in Figs. 4-6. Since values for ttxe control plants were constant and low, they were not plotted. With a dose of 2,500 r (Fig. 4), the various mutation rate curves reached a peak for plants with five tiller groups, dipped for those with six tiller groups, and, except for the apical, rose again. The three curves for "all spikes" (D, E, and F) were similar in shape to the curve of the primary axillary spikes (B). At 10,000 r, the three "all spikes" mutation curves (D, E, and F) and the primary axillary spike curve (B) showed a similar pattern (Fig. 5). There was a peak for plants with four tiller groups, a valley for those with five and a sharp rise for those with six tiller groups. T h e apical spike curve (A) remained nearly constant but decreased for plants with six tiller groups and slightly increased for those with seven. In the 15,000 r treatment (Fig. 6), the three mutation rate curves for "all spikes" (D, E, and F) produced peaks for plants with five tiller groups and decreased for those with six. The p r i m a r y axillary spike mutation curve showed a steady increase for plants with more than three tillers. Apical spike rates reached a peak in plants with three tiller groups and then decreased. ~44utation frequencies by M 1 spike method When mutation rates were calculated by the M 1 spike method and plotted (Fig. 7), mutation curves for the different spike and tiller groups appeared closer to linearity than those calculated by the M 1 plant method (Fig. 3). Mutation rates for apical spikes calculated by both the M 1 plant and M 1 spike methods (Figs. 3 and 7) are identical (Curve A) when tile scales of the two graphs are reconciled. When computed by the M t spike method, apical spikes (A) kad the

highest mutation rate followed by p r i m a r y axillary spikes (B). The remaining three curves (C, D, and E) were similar to B and approached linearity more closely than any curve calculated by the M 1 plant method (Fig. 3). The analysis of variance comparing the five techniques of computing mutations by the M 1 spike method (apical spikes, primary axillary spikes, all mutants, tiller group mutants, and duplicate mutants excluded) showed a highly significant difference between doses. However, there were no differences among the five techniques of computation and likewise the interaction of techniques of computation x doses was not significant. The C.V. was 21.9 per cent. 3/iutation rates based on tiller origin Mutation rates also were calculated according to the origin of tillers and spikes in a plant. Mutation data for the 10,000 r and 15,000 r treatments were subdivided according to location of nodes (tiller groups) on the M 1 plant and types of spikes within each tiller group

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(Table 7). Mutation rates were calculated both by M a plant and M 1 spike methods for "all spikes" in a tiller group, primary axillary spikes, and secondary spikes within a tiller group. Rates at the upper nodes were not very accurate because the number of plants and spikes decreased as distance from the first node increased. Summary of these data are given for nodes one to four plus the apical, for nodes five and six, and for the whole plant. At 15,000 r, the sixth node was equal to or greater than the second node in the "all spikes" mutation rates. At 10,000 r and 15,000 r the highest rates for the primary spikes were at the seventh and sixth

nodes, respectively. For both doses and techniques of computation, mutation rates decreased at the fourth node. At this node, mutation rates for primary axillary spikes decreased only slightly but those of secondary axillary spikes showed a sharp decrease. I n the 10,000r treatment secondary tiller rates were more comparable to the "all spikes" than to primary tiller rates. At the first three nodes, mutation rates calculated by the three different techniques of computation were approximately the same. Mutation rates for spikes of secondary tillers at the fourth node and above were lower than those for spikes from primary tillers. A similar

100

RELATION OF EMBRYO STRUCTURE TO THE EFFECTS OF X-RAYS

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pattern was seen in the 15,000 r treated plants; however, the three techniques of computation gave equal results only at the first node. The summary of the primary axillary rates show that nodes five and six are higher than nodes one to four plus the apical at both doses. However, when the secondaries are considered, nodes five and six are lower. Also, the secondaries are lower than the primaries. The "all spikes" rates are accordingly affected. Mutation rates by the M 2 seedling method

Mutation rates by the M 2 seedling method were analyzed for "all spikes" and for spikes from apical, primary, and secondary tiller buds (Fig. 8). Mutation curves scored by these different techniques were very similar and

almost linear. An analysis of variance which compared the apical spike rate (A) with that of the primary axillary spike (B) showed no difference between these two techniques of computing. The C.V. was 22.7 per cent. There was also no difference in mutation rate between spikes of the secondary and "all spikes" (C and E). For this comparison, the C.V. was 11-4 per cent. When apical spike, primary axillary spike, and secondary axillary spike rates were analyzed, no difference was found between treatments. Here, the C.V. was 18-4 per cent9 Curves for apical spike mutation rates (A) calculated by the M 1 spike and M 2 seedling methods (Figs. 7 and 8) were very similar. The apical mutation curve by the M e seedling method was usually below the other curves (compare with Figs. 3 and 7).

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102

R E L A T I O N OF EMBRYO S T R U C T U R E T O T H E EFFECTS O F X-RAYS

Table 8. Per cent mutant Ms seedlings in mutant spikes from the apical spikes, from the primary axillary spikes, and from the tiller groups at each node Control Total Ms seedlings in mutant spikes Total Plant Apicals Prim. Ax. 1 tiller group* 2 ,, 3 ,, 4 ,, 5 ,, 6 ,, 7 ,, 8 ,, 9 ,,

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6268 613 2031 1913 1286 1359 554 338 150 46 9

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3403 422 1156 1042 725 668 233 191 87 35

22"2 14.4 23"2 25-0 22"3 22-9 23-6 20.4 24.1 5"7

*Counted from ground level, includes primary axillary spikes.

Segregation of Mutants T h e p e r cent of M2 seedling m u t a n t s in m u t a t e d spikes was d e t e r m i n e d for the t o t a l p l a n t , a p i c a l spikes, p r i m a r y a x i l l a r y spikes, a n d e a c h tiller g r o u p s t a r t i n g at g r o u n d level ( T a b l e 8). I n the 10,000 r a n d 15,000 r t r e a t m e n t s , total p l a n t s segregated a p p r o x i m a t e l y 20 p e r cent m u t a n t s . T h e p e r cent for a p i c a l spikes was lower t h a n for total plants a n d p r i m a r y a x i l l a r y spikes. I n the 2,500 r a n d 10,000 r t r e a t m e n t s segregation values were a b o u t 20 p e r cent for the first five nodes. T h e 15,000 r t r e a t m e n t showed a slightly h i g h e r p e r cent of m u t a n t s in t o t a l p l a n t s a n d in e a c h tiller g r o u p c o m p a r e d w i t h the 10,000 r dose. H o w e v e r , a p i c a l spikes in the 15,000 r t r e a t m e n t h a d fewer m u t a n t s p e r spike. T h e p e r cent of m u t a n t s r e m a i n e d fairly c o n s t a n t from tiller g r o u p to tiller group. DISCUSSION

Depth of Planting E a r l i e r studies (e,16) have shown t h a t in cereal plants, the d e v e l o p m e n t of a x i l l a r y buds a n d f o r m a t i o n of tillers were r e l a t e d to seed p l a n t i n g depths. D e e p e r - p l a n t e d seeds usually p r o d u c e d

a n e l o n g a t e d first i n t e r n o d e a n d sometimes a n e l o n g a t e d second i n t e r n o d e . I n wheat,(16) the first three d e v e l o p e d a x i l l a r y buds from seeds p l a n t e d 3 in. d e e p r e m a i n e d d o r m a n t or g a v e rise to w e a k shoots. T h e first b u d to p r o d u c e a strong shoot at a t w o - i n c h d e p t h was g e n e r a l l y the t h i r d a x i l l a r y ; a n d at one a n d o n e - h a l f inches, the second a x i l l a r y b u d p r o d u c e d the s t r o n g shoot.(16) I f the s a m e s i t u a t i o n s h o u l d p e r t a i n to barley, d e p t h of p l a n t i n g w o u l d be v e r y i m p o r t a n t as it w o u l d influence d e t e c t i o n of m u t a t i o n s a n d c a l c u l a t i o n of m u t a t i o n rates. I f the first t h r e e a x i l l a r y b u d s were lost because of p l a n t i n g d e p t h , the t h r e e a x i l l a r y meristems in the b u d at the time of i r r a d i a t i o n w o u l d also be lost, a n d all s u b s e q u e n t axiUary buds w o u l d p r o b a b l y arise from the g r o w t h o f the a p i c a l meristem. T h e p r e s e n t investigation shows t h a t low e m e r g e n c e from d e e p e r seed p l a n t i n g s was caused by i n a b i l i t y o f the plants to p e n e t r a t e the friable soil. T h e r o o t systems a p p e a r e d well d e v e l o p e d in these p l a n t s a n d the total l e n g t h of the shoots were sometimes g r e a t e r t h a n necessary to e m e r g e a b o v e the surface. Thus, it a p p e a r e d t h a t seedling stems from d e e p e r

PATRICIA SARVELLA, R. A. NILAN and C. F. KONZAK

103

plantings lacked the necessary stimulus to push segregation m a y therefore be off one node. T h e high amount of tillering at the first leaf in the through the friable soil. T h e r e is no ready explanation for the finding one-inch depth does not make it likely that that a planting depth of three inches retards this node was affected. Since the buds alternate tillering more than a depth of four inches. on different sides of the stem, any other disHowever, the observation is important since it crepancies were easily detected. indicates that tile depth at which seeds are planted has a very great influence on the Tetrazolium chloride studies ontogeny and development of tillers. Tetrazolium chloride (INT) was used to Transplanting seedlings from petri dishes determine which buds of the seedlings were into milk cartons instead of planting the seeds alive sixteen days after seed irradiation and directly into milk cartons also influenced tiller germination. This stain showed that the bud development. Tillering from the buds in the most sensitive to irradiation was located in the axils of the coleoptile decreased in the trans- axil of the coleoptile. Furthermore, as irradiation planted seedlings, whereas transplanting seed- increased the per cent of stained buds decreased. lings into the field had no adverse effects. In these respects, the I N T test corroborated Transplanting seedlings, therefore, did not observations of tiller development and origin a p p e a r to influence tillering except at the under the influence of irradiation. coleoptile node. The Slight difference in tiller Discrepancies between the two studies might development between plants grown in the field be explained in several ways. T h e r e might have and in milk cartons m a y reflect better growing been a reduction of respiration in certain buds conditions in field soil. which would affect staining capacities. This Irradiation of seeds reduced viability of the reduction might arise through poorer growing various seedling buds, especially of the coleoptile conditions in the seedlings grown in petri dishes, and fourth leaf buds which seemed to be the in spite of the addition of nutrient solution. most sensitive. As expected, this decrease in bud Radiation might have also affected the enzyme viability was greatest with the highest dose. system and thus the staining reaction. PetriT h e plantings in milk cartons showed that dish seedlings were not subjected to the rigorous buds in the axils of the coleoptile were most selection of the field-grown and milk-carton affected by adverse conditions--deep planting plants. Buds that did not stain might have had or irradiation. Buds in the axils of the first, some of the underlying tissues alive and would second, and third leaves usually tillered to about have proceeded to grow under natural conditions. the same degree under both conditions. Reduc- However, tetrazolium chloride can be satistion of tillering from the fourth leaf bud, com- factorily used to indicate bud viability. pared with buds of other leaves, might have been eliminated if the plants had continued Analyses of mutation rates to grow. It has been difficult to ascertain the best I n calculating mutation rates by the various method for determining mutation rates. This methods, it is important to know what per cent is because the mature barley embryo, as already of the sensitive buds will not produce tillers. described, has three axillary primordia and Tile normal procedure in mutation experiments possibly a fourth, in addition to the apical is to irradiate the seeds and transplant the meristem. The presence in the irradiated embryo seedlings at a depth of less than 1 in. which of more than one meristem in different stages of permits the greatest n u m b e r of buds to develop. development with different numbers of cells However, in these experiments, only 50 per leads to formation of chimeras and, as a concent of tile coleoptile buds, which were the first sequence, affects the mutation rates. T h e formed, were viable. Because of this, mutations possible exclusion of mutants ill secondary and were detected one node above the cole0ptile later-formed tillers as postulated by the theory in these plants which lacked tile coleoptile tiller of diplontic selection,(s) also must be considered. group. The results of the mutation rates and The lack of tillering at the first node is also an

104

RELATION OF EMBRYO STRUCTURE TO THE EFFECTS OF X-RAYS

important factor as shown in the depth of planting and tetrazolium chloride studies. Node one in this section therefore refers to the first node of the plant to have tillers. In this study, several different methods were tested in order to determine the most satisfactory one for measuring mutation rates after irradiation of the barley seed. These included the M~ plant, M 1 spike and M 2 seedling methods. Within each of these methods, mutation rates were calculated for "all spikes"; for spikes from apical, primary axillary and secondary axillary tillers; and for spikes from the first four tiller groups. The five curves of mutation rates by the M 1 plant method as related to origin of spikes at a node differed from each other (Fig. 3). This was not surprising since the primary axillary mutation rate should include spikes arising from four different meristems in different stages of development, and should, therefore, be four times higher than the rate in the apical spike which is derived from only one meristem. However, only a three-fold increase was obtained. The death of some meristems as demonstrated by tetrazolium chloride and the depth of planting studies could probably account for this discrepancy. The other three mutation curves for "all spikes", secondary axillary spikes and spikes from the first four tiller groups, differed because of the exclusion of various mutants through grouping. Ideally all curves based on the different analyses should coincide, otherwise the origin of every spike would have to be analyzed. For similar reasons, GAUL(9) concluded that the M~ plant method would have a greater error than the M, spike or M 2 seedling methods. Based upon these results our conclusion agrees with Gaul's. When mutation rates were analyzed by the M 1 spike method (Fig. 7), mutation curves were below that for the apical spike. This is different from the M1 plant method and is attributed to the influence of various meristems and to the use of the spike as the basic measuring unit. I n the M~ spike method, upper axillary meristems (nodes) have been accounted for or even over-compensated by the "all spike" and primary axillary spike analyses because the

upper axillary meristems originated from the apical meristem. The curve corresponding closest to linearity with X-ray dose is the analysis using the four tiller groups. BLIXT et al.(3) found that the mutated sector in NI x pea plants was restricted to one branch and that accurate mutation rates should be related to the number of M 1 branch progenies investigated. From our study, since none of the five techniques of computing mutation rates by the M 1 spike method differed significantly, any technique using the M1 spike method is acceptable for calculating rates and is superior to the M 1 plant method. The high amount of tiilering did not appear to affect mutation rates since the analysis excluding all duplicate mutants was not significantly different from the other techniques of computing mutation rates. With the M 2 seedling method, curves for the five different techniques of computing mutation rates are again ahnost the same (Fig. 8). This indicates that any one of the five techniques of analysis could be used to calculate mutation rates. The theory of diplontic selection (s) suggests that primary axillary spike mutation rates may be lower than apical spike rates since later-formed tillers contain fewer mutant seedlings. Contrary to this theory, however, in the 15,000 r treatment spikes from primary axillary tillers showed an increase of mutation rates as the node increased and spikes from secondary axillary tillers in both doses showed in some cases higher mutation rates than the primaries. Mutation rates may be affected by the amount of tillering as well as the origin of spikes within a tiller group. C-AUL(6) stated that in irradiation experiments the mutation rates decreased with increasing number of tillers per plant. He found that later-formed tillers produced lower mutation rates than the first five tillers(6,7) and differences in tillering were reflected by differences in mutation rates.(9) In another experiment using the M 1 spike method,(s) he found no difference in mutation rates with an increasing number of spikes. Furthermore, high doses produced a highly lethal effect, hence, surviving M 1 seedlings in the field have more space to expand and produce more tillers.O.12) Gaul believes that with the resulting higher

PATRICIA SARVELLA, R. A. NILAN and C. F. KONZAK tillering, there is a greater chance for mutations in the seed to be manifest. Apparently this increase would lead to an overestimation of mutation frequencies.(9) I n our study, therefore, rates were correlated with the n u m b e r of tiller groups per plant. Plants from irradiated seeds in the present investigation showed a reduction of surviving M 1 plants (Table 1) and a decrease in the average n u m b e r of tiller groups and spikes per plant (Table 6). This decrease, however, was not so great as reported by GAUL(9) and MAcKEY.(12) Furthermore, with a large n u m b e r of tillers, as in our experiment, the decrease of mutations due to small progeny sizes with increasing doses should not be sufficient to cause an underestimation of the mutation rate as prbposed by GAUL.(9) In support of this contention, plants with more tillers did not show a significant decrease of interchanges, although there was a trend in this direction. When the n u m b e r of tiller groups per plant is studied in relation to the three radiation doses (Figs. 4, 5, and 6), similarities and differences in mutation curves are found to be dependent on the dose, type of spike, and n u m b e r of tiller groups. Apical spike mutation rates were not affected by increased n u m b e r of tiller groups and remained relatively constant. This stability was expected because the number of tiller groups in a plant should not appreciably affect mutation rates in apical spikes unless normal cell competition early in plant development had excluded some m u t a n t cells. Primary axillary spike mutation rates were higher than apical spike rates. These rates were expected to differ by at least a factor of four because of the corresponding four meristems in the seed at irradiation. I f primary axillary spikes exhibited all the mutants present, the primary axillary mutation curves should then coincide with the "all spike" curves. This should also be true according to the theory of diplontic selection. However, the primary axillary spike curves were lower than the "all spike" curves making it seem that the n u m b e r of mutants is affected by the n u m b e r of tillers and that with more tillers, more mutants have the chance to be manifest. Peaks and valleys in mutation curves for the

105

different tiller groups at all three dose levels (Figs. 4, 5, and 6) are difficult to explain. T h e y might be related to differential radiation response of meristems present in the seed at irradiation or to different stages of development. This possibility has been explained partially by analogous phenomena in other plants. Leaves from irradiated corn showed that the degree of chlorophyll sectoring was related to seed anatomy.O, 18) The first leaves were mottled, later leaves were more elongated and striped, and leaves occurring after about the fifth leaf were normal. This pattern agreed with the primordia of the five leaves present in the seed at irradiation. I n tomatoes, there was also a relationship between chlorophyll sectoring in the leaves and seed anatomy.O4) T h e amount of radiation-induced sectoring in the first leaf was m u c h more than in the second leaf and was eliminated in mature leaves. No leaves were differentiated in the tomato seed. Similarly, in barley, leaf chlorophyll deficiencies from irradiated embryos were found to be restricted to one or more of the first four seedling leaves.O~) This n u m b e r is consistent with the number of meristems in the embryo. The peak mutation rate for barley plants from seed treated with 2,500 r (Fig. 4) occurs in plants with five tiller groups. These plants probably are composed of four tiller groups from the four tiller initial cells in the seed and one tiller group from the first initial cell formed as the seedling developed. The decreased mutation rate in plants with six tiller groups m a y be caused by a type of selection related to either a slower rate of division of mutated cells and a faster rate of normal cells (a type of diplontic selection) or to the stage of meristematic development at irradiation. However, the increase in mutation rate in plants with seven tiller groups possibly rules out diplontic selection. At 10,000 r (Fig. 5) the highest point of mutation curves occurs for the fourth-tiller group and the lowest for the fifth-tiller group. This shift of peaks and valleys from the 2,500 r curves m a y be caused by fewer tiller groups per plant, spikes per plant, or seedlings per plant (Table 6). "All spike" curves are m u c h alike in the 2,500 r and 10,000 r doses.

106

RELATION OF EMBRYO STRUCTURE TO THE EFFECTS OF X-RAYS

Mutation curves at 15,000 r for the different tiller groups (Fig. 6) show only one peak for plants with five tiller groups--there is no decline preceding the peak and no second peak. It is possible that the one peak in the 15,000 r dose is actually the second peak, and that elimination of the first peak has occurred partly because the death of some tillers resulted in combining or blending of the tiller groups. Other factors may have resulted in the exclusion of the first peak. Bud sensitivity to irradiation as shown in depth of planting and tetrazolium chloride experiments, and reduction of seedling emergence and survival might be important in the 15,000 r treated plants and may therefore influence the results. Primary axillary spike mutation curves of the three irradiation doses shifted with dose as did the "all spike" curves although in the 15,000 r dose there was no decrease after the peak. Curves for the 10,000 r dose were between those of the other two doses. In contrast to the primary axillary and "all spike" curves, the peak of the apical spike mutation curves shifted from plants with five tiller groups to four, and then to three, as the dose increased. Since peaks and valleys were seen in the mutation curves at all doses, it appears that they are related to the anatomy of the embryo and developing seedling. Relation of mutation rates to node position in plants also has been studied in this investigation (Table 7). The decrease in mutation rates for the fourth node may be because this meristem often was not formed until shortly after irradiation (Fig. 1). After this decrease there was an increase in mutation rate that often was higher than at the first node; whereas in other plants, there was a decrease after the fourth node. The same relationship existed between rates in spikes from secondary tillers compared to primary axillary and "all spike" rates. There seems to be no relation in barley between mutation rates at the first three nodes and the degree of differentiation of the first three meristems at the time of irradiation. STEIN and STErFENSEN(19) also studied the developmental events within the apical meristem of corn by examining the distribution of radiation-induced plant sectoring. They found that the number of mutations is essentially linear

with radiation dose in the first nine leaves. Moreover, the leaves were divided into three groups: (a) those already formed when irradiated, leaves 3-6, (b) those which are part of the apical capital, leaves 7-9, and (c) those formed by the few apical initial cells, leaves 10-11. The latter group showed a linear response to dose up to a point and then reached a plateau which was closely related to the LDs0 in the population examined. It is likely that mutational events in the barley embryo are similar to those in corn and can be likewise subdivided. Mutation rates at upper nodes can be as high as those at the first node. One explanation for high mutation rates at upper nodes is that the plants were permitted to tiller freely. Apparently this greater extent of tillering increased the possibility of detecting a mutant cell. Thus, diplontic selection does not seem to be effective after the initial elimination of the more highly damaged cells. However, when the summarized data and the information of the secondaries at certain nodes is considered there is evidence for this hypothesis. Separating the mutation data by nodes indicates that the summary view may have masked certain effects. The per cent of mutant seedlings within a spike was calculated according to the position of the tiller group. With increasing dose, the per cent of mutations within the mutant spikes increased. This is particularly true for the total plants (Table 8), apical spikes, and to a lesser extent for some of the other tiller groups. When the size of the mutated sector increased with increasing dose, some of the cells of the primordium were probably killed. At 10,000 r and 15,000 r the per cent of mutant seedlings in apical spikes was lower than the 20 per cent observed for spikes of the whole population. This 20 per cent value is the mean segregation frequency of mutants that GAUL(9) used as his constantOS) in calculating the number of ceils in the barley seed meristem at the time of irradiation. Also, it is the same frequency as the interchanges in the apical spikes of the M 1 plants of the present study. The high recovery of mutants in a mutant spike and the relatively constant values within a dose indicate that mutant cells may not be eliminated at later stages in the ontogeny of the M 1 plant.

PATRICIA SARVELLA, R. A. NILAN and C. F. KONZAK SUMMARY

AND CONCLUSIONS

T h e biological, especially genetic, effects of X-rays induced in barley seeds have been investigated to determine the role that embryo structure, node position, depth of planting and degree of M 1 plant tillering play in the development of these effects. The embryo, when irradiated, has three axillary meristems in addition to the apical meristem. The response of each of these initial meristems to radiation has a very great influence on the degree of tillering and the frequency of mutations that will pass through the M a plant. Amount and location of tillering in seedlings was investigated in relation to seed planting depth. As seeds were planted deeper (3-4 in.), survival rate became poorer, and tillering began at higher nodes. I f seeds were transplanted into the ground from petri dishes rather than planted directly, there was a reduction of tillering from the bud in the axil of the coleoptile. Radiation affected the tiller buds similarly. Relation of bud viability to tiUering was determined sixteen days after seed irradiation and germination by means of the vital stain, iodo-nitro-tetrazolium chloride (INT). In seedlings from non-irradiated seeds, buds at the coleoptile node were the least viable. At other nodes a higher per cent of the buds were viable and thus showed a more pronounced effect from irradiation. The second leaf bud was most sensitive to high doses. To determine progress of aberrant cells through the plant from irradiated seed, M 2 seedlings were scored for chlorophyll-deficient mutants. Mutation rates were calculated by the M 1 plant, M 1 spike, and M 2 seedling methods. For each method, mutation rates were calculated for apical spikes, primary axillary spikes, spikes from different tiller groups, and spikes from secondary tillers. O f the three methods used, the mutation rate curves calculated by the M 1 spike method approached closest to linearity with the three X - r a y doses. When plants were used with all primary axillary and apical spikes present and duplicate mutants excluded, this curve was then almost a straight line. Rates for apical spikes and spikes from primary axillary and secondary axillary tillers differed least with the M 1 spike or M 2 seedling methods.

107

This study showed that satisfactory and reliable mutation rates can be calculated from the easily recognized apical spike. Rates also can be analyzed automatically by the M 1 spike method and would not be influenced by death of axillary buds or lack of tillering. The other techniques of computing mutation rates m a y be as useful, but those techniques which utilize spikes from primary axillary and other types of tillers are long and tedious. Anatomy of the mature plant, as well as the seed, was examined in relation to the influence of diplontic selection on mutation rates. Except for a reduction in rates at the fourth node, node location on the M 1 plant did not appear to affect mutation rates. The fourth node normally would be the next one formed after irradiation. Above this node, mutation rates were as high as at the lowest node. Spikes from secondary axillary tillers frequently showed mutation rates as high or higher than those in spikes from primary axillaries or from the "all spike" group. Summarized mutation rates of the primaries at the upper nodes were higher than at the lower nodes. The converse was true tbr the secondaries which also had lower rates than the primaries. When plants were separated by the n u m b e r of tiller groups per plant, mutation rates were generally higher in plants with more tillers. Fluctuations in mutation rate curves were possibly related to meristem development in the embryo and to a radiation-induced decrease in tiller groups, spikes, and seedlings per plant. The most sensitive buds, determined by tetrazolium chloride and depth of planting studies, were located in the axil of the coleopfile and the third or fourth leaf. All these factors could create a masking effect and cause fluctuations in the curves. Frequencies of interchanges in plants with the largest numbers of tillers were as high or higher than those with smaU numbers of tillers. Therefore, diplontic selection, after initial elimination of the most highly damaged cells, does not seem to be as effective as indicated by the summarized data. Essential linearity of mutation rates with increasing tiller n u m b e r is probably related to a greater degree of m u t a n t manifestation in plants with higher numbers of tillers.

108

R E L A T I O N OF EMBRYO S T R U C T U R E T O THE EFFECTS OF X-RAYS

Acknowledgements--The authors would like to thank Dr. OM KAMRA, Mrs. GEORGANNEJOHNSON, Mrs. JENmE RICE, and Mrs. DIANN ROBBERS for their assistance. Also they would like to thank Dr. RUSSELL and Dr. W. J. DRAPALAfor their statistical help: REFERENCES

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