The effect of pollen irradiation on reproductive capacity, seedling growth, and variation of Betula nigra—II. Seedling growth and induced variation

Radialion Botasty, 1973, Vol. 13, pp. 259 to 268. Pergamon Press. Printed in Great Britain.

THE EFFECT OF POLLEN I R R A D I A T I O N ON R E P R O D U C T I V E CAPACITY, SEEDLING G R O W T H , AND V A R I A T I O N OF BETULA NIGRA--II. SEEDLING G R O W T H AND INDUCED V A R I A T I O N KNUD

E. C L A U S E N

Institute of Forest Genetics, North Central Forest Experiment Station, Forest Service, USDA, Rhinelander, Wis. 54501, U.S.A.

(Received 13 July 1973 ; revised) CLAUSEN, KNUD E. The effect of pollen irradiation on refaroductivecapacity, seedlinggrowth, and variation ofBetula n i g r a - - I I . Seedlinggrowth and inclucedvariation. RADIAT:ONBOTANY13, 259--268, 1973.-Betula nigra seedlings were raised from controlled crosses made with pollen exposed to 0, 0.5, I, 2, 4 and 8 kR & g a m m a radiation. Plants fi'om the 8 kR treatment had poorer survival than those fi'om all other treatments and were 11'5 per cent shorter than the controls. Their leaves also averaged 11.0 per cent smaller than those of the control plants but leaf length/width was unchanged. Dry weights of leaves, stems and roots after 8 kR were reduced to 79.7, 72.1 and 62.6 per cent, respectively, of those of the controls. Seedlings from this treatment also had the greatest shoot]root ratio. The 1 kR exposure appeared to have a stimulating effect on plant height, leaf size, and dry matter accumulation, but these increases were not statistically significant. Pollen irradiation had a pronounced effect on seedling variation. Plants with abnormal leaves occm'red at a frequency of 11 "5 per cent in the 8 kR population with asymmetry as the most common type of leaf abnormality. Seedlings with abnormal stems only were rare, whereas plants with various combinations of abnormal leaves and stems increased with the exposure level up to 24.7 per cent after 8 kR. In addition, this treatment level gave rise to 8.2 per cent dwarfs and to 7.7 per cent stunted plants. Thus, the 8 kR exposure resulted in 53-3 per cent aberrant plants, indicating that pollen irradiation should be an effective method of inducing and increasing variation in this species: seedlings h a d r e d u c e d g r o w t h a n d a b n o r m a l INTRODUCTION POLLEN i r r a d i a t i o n has b e e n used successfully to leaves. VIDAKOVIO(20) r e p o r t e d increased v a r i a tion in g r o w t h rates of Salix alba L. seedlings i n d u c e cytological a n d m o r p h o l o g i c a l v a r i a t i o n derived from g a m m a - i r r a d i a t e d pollen. in m a n y h e r b a c e o u s angiosperms,(:) b u t has M o s t studies of forest trees have, however, b e e n r a r e l y b e e n used on w o o d y species. I n an early study, VON EULER(s) o b t a i n e d a p p a r e n t chloro- c o n c e r n e d with the m o r e radiosensitive g y m n o p h y l l m u t a n t s after i r r a d i a t i o n of Populus sperms. Thus, ERICKSSON et al.(5) r e p o r t e d 96 p e r tremuloides L. p o l l e n w i t h 0-5 k R a n d I kR. cent a b e r r a n t meiotic divisions after Larb: leptolepis (Sieb. et Zucc.) G o r d . p o l l e n m o t h e r MEROEN a n d STAII:S(:2) n o t e d an increase in cells were exposed to 0.5 kR. A p e a k m u t a t i o n c h r o m o s o m e f r a g m e n t s w i t h exposures up to 5 k R following a c u t e i r r a d i a t i o n of m a l e buds of r a t e for w a x y p o l l e n grains was r e a c h e d after only 90 R. H e i g h t and, to a lesser degree, Quercus alba L. a n d Q. coccinea M u e n c h h . VAAI:a.MA(~7) found t h a t survival p e r c e n t a g e of d i a m e t e r of 3- a n d 4 - y e a r - o l d Pinus silvestris L. Betula pendula R o t h seedlings from crosses m a d e a n d P. nigra Arn. seedlings d e r i v e d f r o m i r r a d i with i r r a d i a t e d p o l l e n decreased with increasing a t e d p o l l e n increased w i t h exposure level u p to 9 k R b u t 10 k R suppressed growth.(:g) V a r i a b i l i t y exposure u p to 3.2 kR. Less t h a n 1 p e r cent o f the 259

260

KNUD E. CLAUSEN

also increased greatly with the exposure. Similar results have been reported for Pseudotsuga menziesii (Mirb.) Franco.(a,16) In an earlier paper( a) it was shown that pollen irradiation had a pronounced detrimental effect on reproductive capacity ofBemla nigra L. Yield of filled seed was reduced in direct proportion to the pollen exposures and only 59.7 per cent of the filled seed from the highest exposure (8 kR) germinated. In addition, non-viable abnormal germinants increased with the exposure to 12-6 per cent at 8 kR. The use &irradiated pollen also increased the fi'equency of cotyledon abnormalities with exposure up to 13.3 per cent after 8 kR and thus led to increased germinant variability. The further growth and development of the seedlings produced from the previously reported work are described in this paper. The results of this study should add to the limited information on effects of pollen irradiation on growth and morphological variation of non-coniferous forest trees. MATERIALS A N D M E T H O D S

Reciprocal crosses were made in the spring of 1966 between two B. nigra trees (3081 and 3082) using pollen exposed to 0, 0.5, 1, 2, 4 and 8 kR of gamma radiation. Details of the procedures used have been described previously.Ia) Seeds from these crosses were tested for germination during the following September-October and developing germinants pricked out in flats while still in the cotyledon stage. Each germinant was planted in a 6 × 6 × 7.5 cm wooden plant-band filled with a mixture (I : 1 : 1 by volume) of soil, sand and peat. Each flat contained 25 seedlings and represented one replication of one pollen treatment. Thus, for the crosses 3082 (female) × 3081 (male), 9 replications or 225 seedlings were planted from each treatment, if possible. Due to low seed yield, only 16 seedlings were available in one replication of the 4 kR treatment. Because yield of filled seeds and seed germination of crosses from the 8 kR pollen exposure were Iow,(a) insufficient seedlings were produced from most replications. Therefore, only 199 seedlings were planted from this treatment. Due to technical problems, fewer seedlings were produced from the reciprocal crosses and the incomplete data could not be analyzed statistically. Results of

these crosses (3081 × 3082) will, therefore, not be reported in detail in this paper. The seedlings were raised in a greenhouse at a temperature of about 24°C and under a daylength extended by fluorescent and incandescent lamps to 20 h. At regular intervals, the seedlings were fertilized with water-soluble commercial fertilizer (12 : 31 : 14) in order to maintain plant vigor. Fifteen weeks (105 days) after planting, seedling survival and f,'equency of abnormalities were recorded and samples of the seedlings were measured and harvested for dry weight determinations. These samples included only normalappearing seedlings (excluding forked plants) and for most replications consisted of 15 seedlings. I-Iowever, due to the high proportion of abnormal plants in certain flats, particularly in crosses from the 8 kR treatment, only 10 plants were sampled in these flats. Measurements included : height to leaf number 5, total plant height, number of leaves, blade length and width (at its widest point) of leaf number 5. Following measurements, the sample plants were removed from the flats. The growing medium was carefully washed from the roots and the plants were allowed to airdry. Each sample was then divided into leaves, stems, and roots and the oven-dry weight & e a c h component was determined on a per seedling basis. The results were analyzed statistically on the basis of sample means using analysis of variance and Duncan's new multiple range test for comparison of treatment means.(~a) As the seedlings developed, those showing abnormalities in growth habit and in leaf or stem morphology were marked with appropriate labels. After 105 days each abnormal seedling noted was classified into one of the following 5 groups: abnormal leaves only, abnormal stem only, abnormal leaves and stem, dwarf plant with 5 or more small leaves, stunted plant with 5 or less normal leaves. The last class consisted mostly of plants with reduced height growth, but otherwise of normal appearance. RESULTS

Survival A few seedlings from the control and each of the 4 lower pollen exposure levels died giving an average survival of 98"8 per cent for the 0-4 kR

EFFECT OF POLLEN IRRADIATION ON BETULA NIGRA--II. treatments. Only seedlings from the 8 kR treatment had a significantly lower survival at the 1 per cent level (91.1 per cent). Thus, the plants produced from pollen exposed to 8 kR apparently were weaker than those fi'om the other treatments. This was verified as the plants from this treatment grew older. By June 1973, when the plants were 6 yr old, 7.1 per cent of the plants originally classified as normal had died. In addition, 37.5 per cent of the plants with various abnormalities had died and 10.0 per cent of them were very weak. Growth Seedlings from the different pollen exposures varied little in height to leaf 5 and in number of leaves (Table 1). Those from the 1 kR treatment were the tallest, while those from the 8 kR treatment were the shortest (P~0.05). Differences among seedlings fi'om other treatments were not significant (Table 1). I f the stunted plants had been included in the measurements, the 8 kit treatment could have been shown to have a greater depressing effect on average seedling height. Leaf size of the plants from the 0.5-4 kR treatments varied little from that of the controls, but those fi'om the 8 kR treatment had significantly smaller leaves (P~0.05). Leaf shape, however, as indicated by the leaf length/width ratio, remained essentially unchanged in the normal-appearing plants (Table 1). Foliage, stem and shoot dry weights of seedlings from 'the 1 kR treatment were greater than those from all other treatments, and seedlings from the 8 kR treatment had the lowest dry weights of all groups (Table 2). The effect of this level &pollen exposure was to reduce the weights & t h e aerial plant parts to between 72.1 and 79.7 per cent of the controls (Fig. 1). Root dry weights of seedlings from the 4 lower pollen exposures exceeded that of the control, but these differences were not significant (Table 2). The root weight &seedlings from the 8 kR treatment was significantly lower than that from all other treatment levels (P~0.01) and was reduced to 62.6 per cent of the control (Fig. 1). Thus, the greatest decrease in dry matter production due to pollen irradiation was in the roots. The proportionally greater dry weight

261

decrease in roots than in other plant parts at 8 kR resulted in a shoot/root ratio 17"4 per cent greater than in the controls (Fig. 1), but the difference was not statistically significant (Table 2). Percentage dry weight in leaves, stem, and roots of plants from the 0-4 kR exposure levels averaged 47.0, 22.6 and 30.4 per cent, respectively, and varied little among these treatments. Comparable figures for the 8 kR treatment were 52.3, 22.2 and 25.5 per cent. Thus, at this level a greater proportion of the dry matter was found in the leaves, while that in the stems was essentially unchanged. This may mean that seedlings from the 8 kR pollen exposure were slower to develop than those from the other treatments. As GORDON(8)has shown for seedlings of a related species, B. alleghaniensis Brim, dry matter accumulation in roots increases during the growing season at the expense of that in the leaves, while stem weight remains fairly constant. Although too few seedlings were produced fi'om the reciprocal crosses (3081 ×3082) for valid statistical analyses, the results will be summarized for comparison with the above data. Differences among exposure levels in seedling height, number of leaves and leaf size were too small to show any definite pattern of response. Seedlings from the 8 kR treatment tended to have proportionately broader leaves than those from all other treatments. In dry weigh~t of plant parts, the reciprocal crosses were similar to the 3082 × 3081 crosses except that the seedlings with the greatest stem, shoot and root weights were from the 0.5 kR rather than the 1 kR treatment. The weights of the aerial plant parts after 8 kR were reduced to between 64.9 and 68.6 per cent of the controls, or 5-12 per cent more than in the 3082×3081 crosses. On the other hand, root weight at this level showed a smaller reduction-to 69"0 per cent of the control. Whether these values represent real differences between reciprocal crosses is uncertain, due to the small number of seedlings at certain treatment levels. Variation A large number of seedlings showed abnormalities in leaf and stem characteristics or in growth habit (Table 3). Plants with abnormal leaves only occurred at all pollen exposure levels, except the 1 kR level but only in the 8 kR

262

K N U D E. CLAUSEN

6 6 6 6 6 6 u

oI 0

,

r~

u-)

,.cl u"

0

~

~

N~N~Ng 0

~

0

~

~

6 6 6 6 6 6

0

Ill

0

EFFECT OF POLLEN I R R A D I A T I O N ON BETULA JVIGRA--II.

263

Table 2. Average oven-dry weight per seedling of plant parts and shoot/root ratio at 105 days Pollen exposure, kR

Stem weight,J mg

Shoot weight,* mg

Root weight,J" mg

Shoot] Root*

n

Foliage weight,* mg

0

130

177.5±11.49ab

83"3+5.05 ab

260.84-16.13 ab

109.84- 8.19a

2.41-t-0-119 ab

0.5 1

130 135

168-5+ 7.78ab 193.44-16.69 a

83.5-t-4.21 ab 96.04-9.19 a

252"0+I1.05ab 289"44-25.43 a

116.6,-!-5-07a 125.14-10.12 a

2.174-0.066 b 2.324-0.119 b

2

135

182-24-18.48 ab

88'44-8.23 ab

270"74-26.54 a

121.34- 7.49 a

2"224-0.139 b

4 8

130 100

185-24-14-67 ab 141.44-21.02 b

84.04-7.61 ab 60"14-8.88 b

269"24-22.08 ab 201.54-29.82 b

114.94-12.00 a 68.84- 7.55 b

2-444-0.159 ab 2.824-0.199a

*Values followed by the same letter are not statistically different from each other at the 5 per cent level of significance. "~Values followed by the same letter are not statistically different from each other at the 1 per cent level of significance.

II0I -~

'°°l-

I 80

o Stem weigh,* .Shoot wei;hfNs"

70

D Root w e i g h t ~

~

•

N ~ K~'~

Shoot/Root*

60-

o

I

I

500 I000

I

I

2000

4000 Exposure,

I

8000

R

Fxo. I. Effect of pollen irradiation on dry matter accumulation in Betula nigra seedlings. Key to differences among exposure levels: N.S. = not significant, * = significant at P ~< 0.05, • * = significant at P ~< 0.01. p o p u l a t i o n was the frequency of these plants (11.5 per cent) significantly greater t h a n i n the control p o p u l a t i o n . Seedlings h a v i n g a b n o r m a l stems w i t h o u t a c c o m p a n y i n g leaf abnormalities were r a r e - - a frequency of only 1.1 per cent at the highest t r e a t m e n t level ( T a b l e 3). Plants with various c o m b i n a t i o n s of a b n o r m a l leaves a n d stems occurred after exposures to 1-8 k R a n d

were the most f r e q u e n t class of aberrants. T h e frequency of these a b e r r a n t seedlings increased with the exposure level u p to 24.7 per cent after 8 k R b u t was only significantly different from that i n the controls at the highest t r e a t m e n t level. T h e 8 k R exposure also gave rise to 8.2 per cent d w a r f plants with 5 or more small leaves a n d to 7.7 per cent stunted b u t otherwise n o r m a l plants

264

K N U D E. CLAUSEN

w i t h 5 or less leaves. Thus, the highest p o l l e n exposure used in this s t u d y resulted in 53.3 p e r cent a b e r r a n t p l a n t s ( T a b l e 3). T h i r t e e n b r o a d classes of l e a f a b n o r m a l i t i e s ( T a b l e 4) a n d 7 stem a b n o r m a l i t y classes ( T a b l e

5) were r e c o g n i z e d in this study, i n d i c a t i n g that a g r e a t a m o u n t o f s e e d l i n g v a r i a t i o n was i n d u c e d in B. nigra b y pollen i r r a d i a t i o n . Selected seedlings with various c o m b i n a t i o n s of leaf a n d stem a b n o r m a l i t i e s are shown in Fig. 2.

Table 3..Number and type of abnormal seedlings appearing hz Betula nigra crosses made with irradiatedpollen Pollen exposure, kR

n

Abnormal leaves

Abnormal stem

Abnormal leaves and stem

Dwarf plants

Stunted plants

Total no. aberrant plants

0 0.5 1 2 4 8

223 222 220 222 215 182

3 1 0 2 2 21"

0 0 0 0 0 2

0 0 1 4 7 45*

0 0 0 0 3 15"

0 0 0 0 0 14"

3 1 1 6 12 97*

Total

1284

29

2

57

18

14

120

*The number of abnormal seedlings at this ta'eatment level is significantly greater (P~0"01) than that at all other treatment levels according to tables by KASTENBAUMand BOWMAN.(10)

Table 4. Frequencyof leaf abnormalities observedin Betula nigra crosses made with irradiatedpollen Leaf abnormality class* 7 8 9 10

Seedling type

1

2

3

4

5

6

Abnormal leaves only Abnormal leaves and stem

0

0

0

2

9

2

13

2

1

2

12

9

1

10

2

17

2

Total

2

12

9

3

19

4

30

4

* Type of abnormality Size and/or shape

Margin

Other

Class code 1 2 3 4 5 6 7 8 9 10 11 12 13

11

12

13

2

0

0

1

32

6

5

5

1

0

72

7

7

5

I

1

104

Description minute leaves very small leaves small, narrow, irregular leaves basal leaves crowded, semi-entire long and narrow leaves broad leaves with rounded base leaves asymmetric asymmetrical and twisted leaves semi-entire simply serrate crenate variegated leaves some leaves opposite

Total

FIG. 2. Fifteen-weeks-old Betulu

nigra seedlings produced with irradiated pollen and showing various combinations of leaf and stem abnormalities (classified as in Tables 4 and 5). Ruler is 15 cm long. (a) 4 k R exposure, left to right: 4-5-D, 3-A, 2-12-B, Normal, 11-B. The variegated seedling (center) originally had 3 yellow-green cotyledons. (bj 8 k R exposure, left to right: 4-11-B, 7, Normal, 2-B-F, 5-7-D. (c) 8 k R exposure, left to right: 6-10, 2-9-A, Normal, 7-10-C 7-B-E. Seedling No. 2 originally had fused cotyledons; No. 4 had 4 partially fused cotyledons.

EFFECT OF POLLEN I R R A D I A T I O N ON B E T U L A NIGRA--II.

265

Table 5. Frequency of stem abnormalities observed in Betula nigra crosses made with irradialed pollen Stem abnormality class* C D E F

G

Total

2 29

0 4

0 5

0 1

0 1

0 1

2 60

31

4

5

1

1

1

62

Seedling type

A

B

Abnormal stem only Abnormal leaves and stem

0 19

Total

19

* Type ofabnormali~ Size

Class code A B C D E F G

Form Color Oflaer

Description little or no stem elongation short internodes thin and weak stem basal crook fasciation no anthocyanin present twisted basal branch

T h e most c o m m o n l e a f a b n o r m a l i t y observed in plants with a b n o r m a l leaves only a n d in those having b o t h l e a f a n d stem a b n o r m a l i t i e s was l e a f a s y m m e t r y ( T a b l e 4). L o n g a n d n a r r o w leaves were also f r e q u e n t as were v e r y small leaves. Short internodes was the most c o m m o n class of stem a b n o r m a l i t y , b u t p l a n t s with little or no stem e l o n g a t i o n were also frequent ( T a b l e 5). T h e p l a n t s with b o t h l e a f a n d stem a b n o r m a l i t i e s showed 27 different c o m b i n a t i o n s of the 13 leaf a n d the 7 stem a b n o r m a l i t y classes. T h e various c o m b i n a t i o n s a r e too n u m e r o u s to discuss in detail. H o w e v e r , most of the plants in this g r o u p h a d 1 leaf plus 1 stem a b n o r m a l i t y , while

those with 2 leaf a b n o r m a l i t i e s a n d 1 stem a b n o r m a l i t y were less c o m m o n . I n only 2 p l a n t s were 1 leaf a n d 2 stem a b n o r m a l i t i e s c o m b i n e d , a n d j u s t 1 p l a n t h a d 2 l e a f plus 2 stem a b n o r m a l i t i e s ( T a b l e 6). T h e frequency of seedling a b n o r m a l i t i e s in the reciprocal crosses was o n l y a b o u t o n e - h a l f as large as that in the 3 0 8 2 × 3 0 8 1 crosses b u t followed a similar p a t t e r n . Thus, seedlings with a b n o r m a l leaves only increased w i t h increasing exposure levels up to 7.2 p e r cent after 8 kR. T h e r e were no plants with a b n o r m a l stems only, b u t at the 8 k R level 14.5 p e r cent o f the p l a n t s h a d b o t h a b n o r m a l stems a n d leaves. Similarly,

Table 6. Frequency of seedlings with different numbers of leaf and stem abnormalities

Pollen exposure, kR

Number and type of abnormalities I leaf 2 leaf 1 leaf and and and 2 leaf 1 stem 1 stem 1 stem 2 stem

2 leaf and 2 stem

Total

n

1 leaf

0 0.5 I 2 4 8

223 222 220 222 215 182

3 1 0 2 2 18

0 0 0 0 0 3

0 0 0 0 0 2

0 0 0 4 5 31

0 0 1 0 2 11

0 0 0 0 0 2

0 0 0 0 0 1

3 1 1 6 9 68

Total

1284

26

3

2

40

14

2

1

88

266

KNUD E. CLAUSEN

only 2-9 per cent of the plants at this treatment level were dwarfed plants. Stunted plants, on the other hand, occurred at all treatment levels except the control, but the highest frequency was 4.4 per cent. Most of the plants with combined leaf and stem abnormalities had just 1 abnormality of each class. T h e lower frequencies of seedling abnormalities encountered in the reciprocal crosses m a y be due to differential radiosensitivity of the 2 pollen lots or to interactions between pollen lots and female genotypes, but this could not be demonstrated due to the limited samples. As previously reported, iS) the frequency of aberrant germinants observed in this study was unusually high and increased in direct proportion to the pollen exposure level. For the purpose of comparison with the present data, this relationship is shown in Fig. 3. T h e frequency of aberrant seedlings, however, did not follow the same pattern. I t rose gradually up to 4 kR but then increased steeply to a total of 53.3 per cent at

50 • T o t a l aberrant germinonts o Dwarfs plus stunted plants A P l a n t s with abnormal leaves, s t e m s , o r bo'Ph I:l T o t a l aberrant seedlings

40

50 •

rtel

1.•

j.J ~,S"

20

10~

500 1(3OO 2000

~.SSS.,"

4000

Exposure,

80130

R

FIG. 3. Effect of pollen irradiation on frequency of aberrant gerrninants, short seedlings, and seedlings with leaf and/or stem abnormalities in Betula nigra.

8 kR or twice the frequenc 3, of aberrant germinants (Fig. 3). This change, while partly due to the high n u m b e r of short plants (dwarfs and stunted plants) at this treatment level, is primarily due to the striking increase in plants with abnormal leaves, stems or both (Fig. 3). MABucm a n d MATSUMURAill) reported a similar exponential relationship between mutation frequency and exposure level after acute irradiation of maize pollen, while the response was linear for chronic treatments. D I S C U S S I O N

Pollen irradiation had only a moderate effect on B. nigra seedling growth in this study. Plants raised from crosses made with pollen exposed to 8 kR had poorer survival and were shorter than control plants. These results agree with those of SZIKLAI and EL-LAKANY(TM who reported that Pseudotsuga menziesii seedlings from 2 kR treatments were taller than control plants, while 10 kR suppressed height growth. Similarly, height of Salix alba crosses made with g a m m a irradiated pollen was reduced as the exposure level increased from 10 kK to 95 kR.(20) VAAlZAraAilT) found growth ofB. pendula seedlings grown from irradiated seed to be reduced with exposure levels up to 8 kR for X-rays and up to 15 kR for g a m m a rays. Although pollen irradiation in this study initially appeared to cause only a small reduction in plant growth, its effect became more pronounced with increasing plant age. I n J u n e 1973, 10.7 per cent of the by then 6-year-old plants from the 8 k R treatment, originally classified as normal, were still very small, weak plants. An additional 14-3 per cent of the plants from this treatment showed some growth inhibition. I n this study, seedlings from the 8 k R treatment also had significantly smaller leaves. VAARAMA,(17) on the other hand, reported that leaf length in B. pendula increased at 5 kR, showed no change at 10 kR and was reduced at 15 kR. However, because seed irradiation has a greater effect on seedling growth than pollen irradiation, his results are not directly comparable with those of the present study. Although dry weights of leaves, stems and roots after 8 k R were reduced in this study, only root weight was significantly lower than that of the control.

EFFECT OF POLLEN IRRADIATION ON BETULA NIGRA--II.

267

The effects of pollen irradiation on seedling pollen grains to be nonfunctional. Consequently, variation were much more pronounced. Plants the proportion of normal pollen grains to affected with abnormal leaves, stems or both, showed a but functional grains would be much higher than striking increase after the 8 kR pollen exposure. at the 0.4 kR exposure. In addition, 8.2 per cent dwarfs and 7.7 per cent The high frequencies of germinant and stunted plants occurred after this treatnaent seedling abnormalities observed in this study which, therefore, resulted in a total of 53.3 per may indicate that these parent trees are highly cent aberrant plants. heterozygous or possibly that mutations have M a n y of the abnormalities in leaf and stein been induced. The frequency ofnatural mutation morphology or in growth habit observed in this in B. nigra is unknown, but as indicated by the study are similar to those reported by variation observed in the control plants, it is V~RAMAO 7) after X - r a y and g a m m a irradiation probably low. Inviable abnormal germinants, of B. pendula seeds. He also found asymmetry to probably a type of semi-lethals, occurred at a be the most common leaf abnormality, but frequency of 1.2 per cent and 0"2 per cent of the bifurcated leaves and leaves with chlorophyll germinants had abnormal cotyledons. (3) In deficiencies or irregular shape were common as addition, 1.3 per cent of the seedlings had welh Irregularly branched, bushy and prostrate abnormal leaves. Thus, the spontaneous mutaplants were also noted. Similarly, SCHOLZ(1S) tion frequency in this species m a y be about 1 per obtained chlorophyll mutants and branching cent. Although various mutant forms of the mutants when seeds orB. pendula and B. pubescens European birches are known and have been Ehrh. were treated with X-rays. Seed irradiation utilized for ornamental purposes for m a n y is known to be an efficient means of inducing years, there is little information on actual mutation in various plant species, but the large frequencies. Different cut-leaved and smallamount of variation obtained in B. nigra after leaved types occur occasionally in B. pendula and more rarely in B. pubescens.O) Forms with purple pollen irradiation was unexpected. Pollen irradiation has generally not produced leaves or with fastigiate or 'weeping' growth as high frequencies of seedling abnormalities or habit are also common in cultivation. STERN (15) mutations as those reported in this study. found seedlings ofB. pendula that had flower buds MABUCHI and MATSUMURA,(11) however, found during their second year in the nursery to occur acute irradiation of maize pollen to produce up at a frequency of 1-2 in 104 plants. Recently, to 72 per cent endosperm mutations, and to be a VALANNE and VALANNE(18) located one seedling with variegated leaves among 103 plants raised more effective mutagen than chronic irradiation. BROCK and FRANKLIN, (~) o n the other hand, in Finland from open-pollinated seed of one reported no significant difference in mutation B. pubescens tree. These frequencies are considerrate between acute and chronic treatments of ably lower than those reported here but are, of tomato pollen. High mutation rates after g a m m a course, from other species and probably not irradiation of cotton pollen were also noted by directly comparable. Some of the data from this study indicate that GALEN and ENDRIZZL(7) These authors reported that 84 and 58 per cent of the plants had chromo- the lower exposure levels m a y have had a some aberrations after 0.4 kR and 1.2 kR stimulating effect on early seedling growth. respectively. Frequency of mutations at indivi- Seedlings from the 0"5-2 kR exposures had dual loci ranged from 2-5 to 8.8 per cent at the greater heights to leaf 5 than the control, and 0.4 kR level and from 0-9 to 3.9 per cent at the those from the 1-4 k R treatments were heavier 1.2 kR level. Overall, about 44 and 18 per cent than the control plants. Seedlings from the 1 kR exposure were the tallest, had the biggest leaves of the plants, respectively, showed mutations. Thus, the 0.4 k R treatment appeared to produce and were the heaviest, but these differences were more chromosomal changes and mutations than not statistically significant so the apparent the 1.2 k R treatment. GALZN and ENDRIZZI,(7)as stimulatory effect of this level of irradiation a possible explanation of these results, suggested could not be established with certainty. T h e high frequency of seedlings with abnormal that the 1.2 k R exposure caused most of the

268

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m o r p h o l o g y or g r o w t h h a b i t a n d the wide a r r a y of l e a f a n d stem a b n o r m a l i t i e s p r o d u c e d , p r o v i d e evidence t h a t pollen i r r a d i a t i o n should be a n effective m e t h o d of i n d u c i n g a n d increasing v a r i a t i o n in B. nigra.

Acknowledgement--The author thanks Dr. Tno,'aAs D. RUDOLPH for advice on exposure levels and for irradiating the pollen. REFERENCES 1. BREWBAKER J. L. and EMERY G. C. (1962) Pollen radiobotany. Radiation Botany 1, 101-154. 2. BROCK R. D. and FRA_NKL:NI. R. (1966) The effect of desiccation, storage and radiation intensity on mutation rate in tomato pollen. Radiation Botany 6, 171-179. 3. CLAUSBNK. E. (1973) The effect of pollen irradiation on reproductive capacity, seedling growth, and variation of Betula nigra--I. Seed yield, germination, and germinant abnormalities. Radiation Botany 13, 47-54. 4. EL-LAro,NY M. H. and SZIKLAI0 . (1970) Effects of gamma-irradiation on pollen, on seed, and on seedling characteristics in Douglas-fir. Adv. Front. Plant Sci. 25, 41-51. 5. EmCKSSON G., EKBERO I., EHRENBERC L. and BEVXLACQUAB. (1966) Genetic changes induced by semi-acute gamma-irradiation of pollen mother cells in Larix leptolepis (Sieb. et Zucc.) Gord. Hereditas 55, 213-226. 6. EULER H. YON (1949) Biochemische Untersuchungen an diploiden und triploiden Espen aus normalen und r6ntgenbestrahlten Pollen. Ark. Kemi, 3¢in. Geol. 26(30), 1-19. 7. GALEND. F. and ENDRIZZtJ. E. (1968) Induction ofmonosomes and mutations in cotton by gamma irradiation of pollen. J . Heredity 59, 343-346. 8. GORDONJ. C. (1969) Effect of shade on photosynthesis and dry weight distribution in yellow

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