Chromosoma (Berl.) 15, 123--131 (1964)

From the Welsh Plant Breeding Station, Aberystwyth

CHROMOSOME MOSAICS I N S Y N T H E T I C A M P I t l P L O I D S I N T H E A V E N A E

By

I t . THOMAS and W. T. H. P ~ G ~ I ~ *

With 2 Figures in the Text

(Received December 13, 1963)

Introduction The presence oi cells with different chromosome numbers wi thin the

same p lan t has been recorded in m a n y diverse genera of plants. They are chiefly found in p lants derived from interspecies crosses and part i- cularly following colchicine t rea tment . Such a mix ture of amphiploid cells including those with reduced chromosome complement wi thin the same t issue have been described by FAN~trAVS~ (1945) as "complex chromosome mosaics". The frequency of occurrence of these chromo-

some-deficient cells is generally low, al though up to 15 % aber ran t cells in the Tri t ic inae have been reported by SACKS (1952).

Dur ing studies on synthet ic amphiploids derived from certain te traploid • diploid species-combinations in the Avenae, mosaic cells were often recorded, and the observations on these abnormali t ies form the substance of this paper.

Materials and Methods Sterile hybrids derived from tetraploid X diploid species crosses were treated

with colchicine, using the capping and injection technique described by BELL (1950). The following species were successfully doubled to form partly fertile hexaploid synthetic amphiploids:

A. abyssinica HOe~ST. (2n ~ 28) • A. ~trigosa Sem~],3~. (2n ~ 14) var. S. 75 A. abyssinica ~OCI~ST (2n ~ 28) X A. strigosa S e m ~ . (2n = 14) var. S. 171 A. abyssinica HOC~ST. (2n = 28) • A. brevis 1%OTH (2n = 14) A. barbara PoTT (2n ~ 28) X F 3 se]. A. strigosa subsp, hirtuIa (LAG.) MALZ.

X A. brevis RoT~ (2n ~ 14) A. barbata POTT (2n = 28) • A. longiglumis DuI~. (2n ~ 14) A. barbata POTT (2n = 28) • A. strigosa subsp, hirtula (LAG.) MALZ. (2n = 14) The amphiploid seeds obtained from the colchicine-treated F 1 plants were

sown in pots in a cool glasshouse and the infloreseences fixed and stored in Carnoy solution to which was added ferric chloride for cytological study. The pollen mother cells were studied as smear preparations stained in 2% aceto-carmine solution. Chromosome counts as well as pairing studies were made ~t metaphase-I, and in each instance 20--30 cells were analysed. In addition the progenies of two further synthetic amphiploids were investigated, A. abyssinica ( 2 n : 28)x A. strigosa (2n = 14) Cc 4387 obtained from Dr. F. ZILH~SKu Cereal Division, Ottawa, and Cc 4435 from Professor R. L. S~A~DS, Wisconsin, U.S.A.

* Pressent adress: N. 1%. C., Tengeru, [Box 3101, Arusha, Tanganyika. Chromosoma (BerI.), B4. 15 9

124 H. T~oMAs and W. T. H. PEREGRINE :

Results

Frequency o / m o s a i c cells

I n p l an t s where mosa ic cells were r e c o r d e d t he i r f r e q u e n c y was

1 to 5 pe r an the r , or a p p r o x i m a t e l y 1% to 5% of t h e t o t a l cells (Table l ) ,

f igure wh ich is c o m p a r a b l e to t h a t r e p o r t e d b y SAc~s (1952).

Table 1. The/requency o] mosaic cells at metaphase-I o] meiosis in 6 • amphiploids

A. abyssinica • A. strigosa (S. 75) . . A. abyssinica • A. strigosa (S. 171) A. barbata • A. longiglumis . . . . . A. barbata • A. strigosa subsp, hirtula . A. abyssinica • A. strigosa Cc 4435 . . A. abyssinica • A. strigosa Cc 4387 . .

Number of

Plants Cells examined examined

44 889 1~ 389

158 1~ 264

143 7 137

Percentage cells with reduced chromosome

number

2.02 1.79 0.0 0.38

26.50 15.30

I n t h e s y n t h e t i c a m p h i p l o i d s d e r i v e d f r o m t h e crosses i n v o l v i n g

A . abyssinica a n d A . strigosa t h e n u m b e r of c h r o m o s o m e - d e f i c i e n t cells

I

/ /

• I

"

~ 5

o z L/ 6 8 /o /2 z/ lyO, of chPomosome~ de~klknl per cell

Fig. 1. Frequency of cells with deficient chromosome numbers and the calculated Foisson distributio~

r e c o r d e d seems to be d e p e n d e n t on t h e g e n o t y p e s of t h e species used as

p a r e n t s in t h e in i t i a l F 1 hyb r id . T h e a m p h i p l o i d s p r o d u c e d b y ZILLINSKY a n d S~A~DS h a d a v e r y

h igh f r e q u e n c y of c h r o m o s o m e - d e f i c i e n t cells, m a i n l y due to t h e t w o

Chromosome mosMcs in Avenae amlohiploids 125

p lan t s Cc 4387/1 and Cc 4435/1, which respec t ive ly had 68.33% and 70.37% chromosome-def ic ient cells. These two amph ip lo id p lan t s h a d pol len mo the r cells wi th chromosome numbers ranging f rom 21 to 42 wi thin the same anther , and t h e y were the least fert i le of a n y in t h e series, hav ing a seed set of only 2 to 5% (G~I~ITI tS et al. 1959).

The f requency d i s t r ibu t ion of the combined d a t a for these two p lan t s is represen ted in Fig. 1, t oge the r wi th the ca lcula ted Poisson d i s t r ibu t ion based on a mean chromosome deficiency per cell of 2.76.

Table 2. The [requency o] mosaics at metaphase-I o/meiosis in the A~ generation o/synthetic amphiploids

A. abyssinica • A. strigosa (S. 75) . A. abyssinica • A. strigosa (S. 171) . . A. abyssinica • A. brevis . . . . . . A. barbata • A. longiglumis . . . . . A. barbata x Fa sel. (A. strigosa subsp.

hirtula) X A. brevis . . . . . . . .

N u m b e r of

Plants Cells examined examined

117 2138 30 501 60 1057 15 285

43 763

Percentage cells with reduced c h r o m o s o m e

n u m b e r

1.49 0.79 0.94 0.35

2.22

The calculated variance of 72.6 for a mean of 2.76 indicates that the distribution of chromosome deficiency is not Poisson, showing that there was a tendency for more than one chromosome to be lost.

Chromosome mosaics were also recorded for the A 2 generation of the same amphiploids (Table 2), although the percentage of aberrant cells is less t h a n t h a t found in the A 1 gene- ra t ion.

I n all the anthers conta in ing chromosome- deficient cells the chro- mosome number wi th the h ighest f requency of occurrence was recog- nised as the somat ic number of t h a t p lant . The first and second genera t ion p lan t s of the

Table 3. Per cent chromosome-deficient cells in plants di//ering in somatic chromosome number

Generation

First: A s

Second:A S

C h r o m o s o m e n u m b e r of

p l a n t 2 n =

43 42 41 40

43 42 41 40

T o t a l cel ls e x a m i n e d

157 748 310 207

307 2540

743 470

P e r cent m o s a i c cells

1.91 1.60 2.90 0.00

2.93 0.86 0.81 3.40

syn the t ic amphip lo ids were found to have numbers ranging f rom 40 to 44 chromosomes. I t will be observed from Table 3 t h a t the occurrence of mosaic cells was not associa ted with the basic chromosome n u m b e r of the p lant , as chromosome-def ic ient cells were found in p lan t s differing in somat ic chromosome number .

9*

126 H. TI~OMAS and W. T. H. PXREG~I~n :

Chromosome p a i r i n g in de / ic ien t cells

GRIFFITHS et al. (1959) have described in detail chromosome pair ing in synthet ic amphiploids in the A venae . A study of chromosome pair ing

in cells with the hexaploid chromosome number and in deficient cells wi thin the same anther , will give a clearer indicat ion of the abi l i ty of mosaic cells to undergo meiosis (Table 4).

Table 4. Chromosome pairing in cells with a reduced chromosome complement in Cc 4387/1 and Cc 4435/1 amphiploids

Chromo- Associations found

2o~ v i v I i v , i i i i i

- - [ !13.10 8--19 42 10.12 0--1 0.18 0--1 1.88 0--3 ..41 0--4 2.35 0--9 41 0.007 0--1 0.15 0--1 1.85 0--4 .54 0 3 13.15 9--17 1.61 0--4 4O 39 0 . ~ 0 - 1 0.71 0 - 2 2.00 0 - 5 . 29 - 10.71 7 - 1 4 3.28 1 - s

0.115 0 - 1 1.38 0 - 3 ~.92 0 - 2 ! 11.46 9 - 1 3 2.08 0 - 6 38 0.13 0--1 0.13 0--1 3.12 1--5 ~.87 1--3 10.12 9--12 1.25 0--3 37 - - 0.20 0 - 1 2.20 1 - 4 ).so 0 4 1 0 . 1 8 1 7 - 1 2 2 . 8 0 2 - 4 25 - - 3.00 1 - ~ ).00 5.00 3 - 5 3.00 1 - 3 22 0.50 0 - 1 ~00 2 - - 2 5.50 4 - 7 3 0 0 2 ~

21 - - - - 4--5 18 0 .~ 0----1 0.33 0--1 1.33 1--2 6.33 1--2 3.00 1--3 1.00 0--2 - - - - 4.66 1.66 0--4

There were no peaks for mul t iva len t format ion at different chromo-

some levels, indicat ing tha t the reduced numbers present a random assor tment of chromosomes. I n the Tr i t i c inae , SACHS (1952) reported a high frequency of un iva len ts in reduced cells. I n the present s tudy,

however, the mean n u m b e r of un iva len ts was comparable with t h a t reported by GRIrFITHS et al. (1959) for the synthet ic amphiploids. Evidence was obta ined from a s tudy of chromosome pairing t ha t aber-

r an t ceils can undergo normal meiosis (Fig. 2).

S u r v i v a l o/ de]icient gametes

An est imate of the survival of chromosome-deficient gametes was readily obta ined by s tudying the chromosome numbers of the 1st and 2nd generat ion ~mphiploids, r andom samples of which plants were

recorded at metaphase- I (Table 5).

T~ble 5. Chromosome numbers o /1s t and 2nd generation amphiploids

Chromosome n u m b e r 2 n = Amphip lo ids Genera t ion

38 39 40 ~1 42 43 44

A. abyssinica • A. strigosa . . . A 1 1 0 9 15 27 7 2 A. abyssiniea • A. strigosa . . . A~ 3 2 33 58 15 10 A. abyssinica • A. brevis . . . A 1 0 0 0 0 10 1 1

�9 0 48 1 1 A. abyssinica • A. brevis . . A 2 5 1 A. barbata • A. hirtula -- brevis A 2 0 1 4 2~ 4 1 A. barbata • A. longiglumis . �9 A1 00 0 4 0 0 A. barbata • A. hirtula . . . . A2 0 1 1 11 0 0

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128 H. TuoMAs and W. T. H. PEREGRINE :

The majori ty of the synthetic amphiploids had the expected chromo- some number of 42. The occurrence of progeny with higher or lower numbers than the expected clearly indicates tha t gametes deficient in chromosome number were functional.

In the second amphiploid generation of A . abyss inica • A . strigosa

only 41.4% of the plants had the normal hexaploid number of 42; anthers containing mosaic cells had some pollen mother cells which had a chromosome number of 21 but the absence of plants deficient for more than 4 chromosomes indicates tha t the gametes derived from such cells were non-functional.

All the progeny derived from crosses involving Cc4387/1 and A . sativa had chromosome numbers of 38, 39 and 40 (Table 6). Two

Table 6 Chromosome number o/ F 1 plants between synthetic amphiploids and natural hexaploids

i Chromosome number 5152_

A. barbata/A, hirtula (6x) • A. byzantina . . . . A. barbata/A, hirtula • A. ]atua . . . . . . . . A. barbata/A, hirtnla • A. nuda . . . . . . . . A. barbata/A, hirtula • A. sativa . . . . . . . . A . barbata/A, hirtula • A. sterilis . . . . . . . A. abyssinica • A. strigosa (S. 171) • A. sativa . A. abyssinica • A. strigosa (Ce4387/1) • A. sativa A. abyssinica • A. brevis • A. sativa . . . . . .

progenies from the cross A. barbata/A, hirtula (6x) • A. byzant ina (6x), and one from the cross A . barbata/A, hirtula • A . / a t u a are of interest in tha t they had chromosome numbers of 47, 51 and 55 respectively.

While the deficiency of four chromosomes can be explained on the basis of irregular disjunction at metaphase-I, it is difficult to explain on a similar basis why crosses of synthetic amphiploids and natural hexap]oids gave progenies with up to eleven chromosomes more than the hexaploid number of 42. No disjunction as irregular as this was observed at metaphase of any of the plants.

A possible explanation is that an unreduced mosaic cell with a chromosome number in excess of 21 was fertilized by a normal gamete of the natural hexaploid. According to this interpretation, the A . barbata/

A . hirtula • A . byzant ina F 1 hybrid with 55 chromosomes could arise from the fusion of an unreduced 34 chromosome cell and a normal gamete of 21 from A . byzant ina.

This is consistent with the data, since the natural hexaploids in- variably have 21 bivalents at first metaphase, and irregular disjunction is rare. I t is clear then tha t gametes with the extra chromosomes must

Chromosome mosaics in Avenae amphiploids 129

have originated with the maternal plant, i.e., in the synthetic amphi- ploid, since this was used as the female in all the crosses studied. I t is of interest to note too tha t all these backerosses are sterile, regardless of chromosome deficiencies or additions.

Discussion

These investigations have shown tha t in synthetic amphiploids of Avenae, pollen mother cells with a reduced chromosome complement occur within the same anther. The frequency of these mosaic cells was dependent on the species used in the original cross, which suggests tha t the abnormali ty occurs in certain gene combinations. Plants showing this phenomenon of mosaieism were also recorded in later generations of the amphiploids, as well as in progenies derived from crosses between synthetic amphiploids and the natural hexaploids.

The occurrence of chromosome-deficient cells in later generations further indicates tha t this phenomenon is genetically controlled. The genetic control of chromosome loss in derivatives of a cross between A. 8ativa and A. byzantina has also been reported by GmFFITHS and TtIOMAS (1954).

SACHS (1952) propounded tha t the occurrence of chromosome defi- cient cells could arise as a result of gene-controlled spindle abnormalities at the mitotic divisions preceding meiosis. DA~Lr~GTO?r and T~OMAS (1937) made a detailed study of meiosis in a Festuca-Lolium derivative, and showed tha t abnormal spindles at meiosis can occur in such a deri- vative. S~IITR (1942) reported a recessive factor which interferes with the normal development of spindle in pollen mother cells of barley. The formation of two separate and independent spindles in one cell has been established as the cause of variation in chromosome number in somatic tissue of Ribes nigrum (VAA~AMA 1949). Genetic control of spindle abnormalities during the pre-meiotie division could account for the occurrence of mosaic cells in the amphiploids. Groups of deficient cells were sometimes found in close association with each other within the same anther; this supported the theory of spindle abnormalities being responsible for the presence of mosaic cells. Furthermore, the presence in later generation amphiploids, and in crosses of these with natural hexaploids offers additional evidence in support of an explanation based on genetically-controlled spindle abnormalities.

No evidence for the oecmTence of mosaics in root-tip tissue was found. I t is difficult to make an accurate assessment of the time at which these mitotic abnormalities occur in somatic tissue but they probably occur at a stage just preceding meiosis, since cells with very reduced chromosome numbers would be too unbalanced to survive for

130 H. T~o~.as and W. T. I-I. PEREGI~rNE :

many generations. Evidence for such an unbalance causing cell degenera- tion is furnished by SNOAD (1955) who reported variation in chromosome number in root-tips and pollen mother cells of Hymenocallis calathinum, the range of chromosome number obtained being much higher in the root-tips than in the pollen mother cells. He concluded that the pre- meiotic cells had probably earned the variation through many cell generations, but that the conditions only permit the larger and more balanced numbers to propagate.

OKSALA (1944) showed that in the dragon-fly Aeschna ]uncea, the precocity of meiosis is gradually developed during at least two preceding mitoses, although none appears before. These pre-meiotie mitoses may differ from other somatic divisions, and spindle abnormalities are more likely to occur at this stage. VAA~AMA (1949) has shown in tetraploid Ribes nigrum that such irregularities during mitosis occur at an earlier stage, even during root-tip divisions. In the Avenae, however, spindle abnormalities have not been recorded as early as this. The fact that these pre-meiotic irregularities do occur in the amphiploid Avenae suggests that the mosaic cells arise at the same time as the spindle abnormalities. This is consistent with the data in Fig. 2 which show that chromosomes were not lost independently of each other, as might be expected during chromatin migration, and this indicates that some cellular defects resulting from spindle abnormalities occurred at a previous division affecting groups of chromosomes.

HUSKIES (1948, 1949) has shown that somatic chromosome pairing and segregation could be induced in the root tips of AIlium cepa by an aqueous sodium nucleate solution. This phenomenon could give rise to cells with reduced chromosome numbers. Sac~s (1952) however, failed to repeat these effects in root tips of T. monococcum. With the paucity of cytological evidence for the natural occurrence of somatic pairing in higher plants it would be unreliable to base an interpretation of the incidence of chromosome-reduced cells on the phenomenon of "somatic meiosis".

Summary 1. Chromosome-deficient cells were found at the meiotic stage in

synthetic amphiploids derived from various diploid-tetraploid combina- tions in the Avenae.

2. Chromosome-deficient cells were found to be dependent on the species used as parents to produce the triploid F 1 hybrid.

3. Chromosome deficiency in two amphiploid plants Cc 4387/1 and Ce 4435/1 did not follow a Poisson distribution.

4. A study of chromosome pairing revealed that aberrant cells undergo normal meiosis.

Chromosome mosaics in Avenae amphiploids 131

5. F i rs t (A1) and second (A2) generat ion amphiploid plants showed

tha t chromosome-deficient gametes were functional .

6. The occurrence of chromosome-deficient cells is a t t r ibu ted to spindle abnormali t ies at the pre-meiotie divisions.

Acknowledgements. We wish to express our thanks to Professor P. T. T~oM~s, Director, Welsh Plant Breeding Station, for his advice and encouragement through- out this study and to Dr. D. J. GI~IFFITtIS for help with the paper while in prepara- tion. We are grateful to Dr. D. G. ROWLANDS for permission to use his data and to Miss IRENE D. I%EES for editorial assistance.

References BELL, G. D. H. : Investigations in Triticinae. I. Colchieine techniques for chromo-

some doubling in interspeeific and intergeneric hybridization. J. agrie. Sci. 49, 9--18 (1950).

DAI~LINGTON, C. D., and P. T. T~o~As: Breakdown of cell division in a Festuca- Lolium derivative. Ann. Bot. (Lond.), N.S. 1, 747--761 (1937).

FA~I(I~AUSER, G.: The effects of changes in chromosome number in amphibian development. Quart. Rev. Biol. 2, 20--78 (1945).

Gt~IF~'rTHS, D. J., D. G. I%OWLA~I)S, and W. T. H. PEI~EGI~I~E: Cytogenetic rela- tionship of certain artificial and natural species of Arena. J. agric. Sci. 52, 189-- 199 (1959).

- - , and P. T. THOI~IAS: Genotypie control of chromosome loss in Avenae. Proc. Ninth Int. Genetic Congr. 1953. Caryologia (Firenze) Suppl. 6, 1172--1175 (1954).

HvsxI~s, C.L.: Chromosome multiplication and reduction in somatic tissue. Nature (Lond.) 161, 80--83 (1948);--The nucleus in development and differ- entiation and the experimental induction of "meiosis". Proc. 8th Int. Genetics Congr. tIereditas (Lurid), Suppl., 274--285 (1949).

OKSaLA, T. : Zytologische Studien an Odonaten. II. Die Entstehung der meiotischen Pr~kozit~t. Ann. Acad. Sci. fenn. A IV 5, 1--33 (1944).

SAcHs, L. : Chromosome mosaics in experimentM amphiploids in the Triticinae. Heredity 6, 157--170 (1952).

S~ITH, L. : Cytogenetics of a factor for multiploid sporocytes in barley. Amer. J. Bot. 29, 451--456 (1942).

S~OaD, B. : Somatic instability of chromosome number in Hymenocallis calathinum. Heredity 9, 129--133 (1955).

VAAnA~A, A.: Spindle abnormalities and variation in chromosome nmnher in 2Ribes nigrum. Hereditas (Lund) 35, 136--162 (1949).

Dr. HUGH TI-IOMAS, Welsh Plant Breeding Station,

Plas Gogerddan, near Aberystwyth, Great Britain