cytological studies in oenothera with special reference to the relation of chromosomes to nucleoli

Cytological Studies in Oenothera with Special Reference to the Relation of Chromosomes toNucleoliAuthor(s): P. N. BhaduriSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 128, No.852 (Feb. 15, 1940), pp. 353-378Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/82166 .

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The chromatic behaviour of the eel The chromatic behaviour of the eel

REFERENCES

De Beer, G. I926 The pituitary body. Oliver and Boyd. Hogben, L. 1923 Quart. J. Exp. Phys. 13.

Hogben, L. and Gordon, C. 1930 J. Exp. Biol. 7, 286.

Hogben, L. and Landgrebe, F. 1939 Proc. Roy. Soc. B, 128, 317.

Hogben, L. and Slome, D. 1931 Proc. Roy. Soc. B, 108, 10. - - 1936 Proc. Roy. Soc. B, 120, 158.

Lode, A. 1890 S.B. Akad. Wiss. Wien, 101, 130. Neill, R. M. 1939 J. Exp. Biol. (in the Press). Odiorne, J. M. I933 Proc. Nat. Acad. Sci., Wash., 19, 329. Stendel, W. I914 Die Hypophysis Cerebri. Oppel. Lehrb. d. Verg. lMikr. Anat. d.

Wirbeltiere, part 8, Jena.

Waring, H. 1938 Proc. Roy. Soc. B, 125, 264.

Cytological studies in Oenothera with special reference to the relation of chromosomes

to nucleoli

BY P. N. BHADURI

King's College, University of London

(Communicated by R. R. Gates, F.R.S.-Received 27 October 1939)

INTRODUCTION

Compared to the vast amount of work done on the cytology of this group of plants very little attention has been paid to a systematic analysis of the somatic chromosomes. The necessity of such a study was felt long ago by de Vries and Boedijn (1923, I924), who tried to analyse the chromosomes of Oenothera Lamarckiana and its several mutants. Since then the somatic chromosomes of Oenothera species have been illustrated from time to time but detailed observations in this connexion remained to be made. Such a situation has probably arisen from the fact that geneticists realized that information from such a study would not throw much light on elucidation of their genetical observations, as all the species of Oenothera showed a stable chromosome number of 2n = 14, without any significant variation in their morphology. Probably a more important factor explaining the dearth of literature on this subject is the technical difficulty involved in getting good cytological preparations of root-tips of Oenothera species.

REFERENCES

De Beer, G. I926 The pituitary body. Oliver and Boyd. Hogben, L. 1923 Quart. J. Exp. Phys. 13.

Hogben, L. and Gordon, C. 1930 J. Exp. Biol. 7, 286.

Hogben, L. and Landgrebe, F. 1939 Proc. Roy. Soc. B, 128, 317.

Hogben, L. and Slome, D. 1931 Proc. Roy. Soc. B, 108, 10. - - 1936 Proc. Roy. Soc. B, 120, 158.

Lode, A. 1890 S.B. Akad. Wiss. Wien, 101, 130. Neill, R. M. 1939 J. Exp. Biol. (in the Press). Odiorne, J. M. I933 Proc. Nat. Acad. Sci., Wash., 19, 329. Stendel, W. I914 Die Hypophysis Cerebri. Oppel. Lehrb. d. Verg. lMikr. Anat. d.

Wirbeltiere, part 8, Jena.

Waring, H. 1938 Proc. Roy. Soc. B, 125, 264.

Cytological studies in Oenothera with special reference to the relation of chromosomes

to nucleoli

BY P. N. BHADURI

King's College, University of London

(Communicated by R. R. Gates, F.R.S.-Received 27 October 1939)

INTRODUCTION

Compared to the vast amount of work done on the cytology of this group of plants very little attention has been paid to a systematic analysis of the somatic chromosomes. The necessity of such a study was felt long ago by de Vries and Boedijn (1923, I924), who tried to analyse the chromosomes of Oenothera Lamarckiana and its several mutants. Since then the somatic chromosomes of Oenothera species have been illustrated from time to time but detailed observations in this connexion remained to be made. Such a situation has probably arisen from the fact that geneticists realized that information from such a study would not throw much light on elucidation of their genetical observations, as all the species of Oenothera showed a stable chromosome number of 2n = 14, without any significant variation in their morphology. Probably a more important factor explaining the dearth of literature on this subject is the technical difficulty involved in getting good cytological preparations of root-tips of Oenothera species.

353 353



P. N. Bhaduri

A new line of research has been opened up since the relation of Sat-chromosomes to nucleoli was discovered by Heitz (I93Ia,b) and Mc- Clintock (I931, I934). The application of their theory regarding the corre-

spondence of the number of nucleoli to the number of Sat-chromosomes is throwing considerable light on the understanding of relationship and

probable origin of some of the different groups of plants. In view of these facts, a study of the somatic chromosomes of Oenothera species in relation to nucleoli was thought extremely desirable. Considerable difficulty has been overcome by the recent discovery of a method described by Semmens and Bhaduri (i939) and Bhaduri (I938), by which the chromosomes and the nucleoli can be stained differentially. The present study is part of a

contemplated exhaustive study of the chromosomes in different species of Oenothera as well as other related genera.

While studying the catenation of a number of species and hybrids of

Oenothera, during the summer of 1938, an attempt was also made to correlate the relations of chromosomes with nucleoli during meiosis as well as during mitosis.

MATERIAL AND METHODS

For a study of somatic chromosomes root-tips were collected from the

following species* and hybrids of Oenothera which were grown in the green- house at the Courtauld Genetical Laboratory, Regent's Park:

(1) O. Hazelae Gates var. parviflora Gates.

(2) 0. ammophiloides Gates and Catcheside.

(3) 0. angustissima Gates var. quebecensis Gates.

(4) Oenothera sp. undescribed, from St Jerome, Quebec. (5) 0. biformiflora Gates var. cruciata Gates.

(6) Oenothera sp. undescribed, from Moose Jaw, Sask.

(7) 0. Lamarclciana de Vries.

(8) 0. blandina de Vries.

(9) 0. Hookeri Torrey and Gray. (10) 0. biformifora var. cruciata x O. Lamarckiana.

(11) 0. missouriensis Sims.t

Root-tips were also collected from seeds, which were germinated on moist cotton-wool in petri dishes and kept near the radiator in the laboratory

* Most of the new species and varieties studied in this paper were described by Gates (1936).

t This is a hybrid of two different strains; one from western North Dakota, with pink spots on the sepals, the other of unknown origin but without pink spots (see Gates 1939).

354



Cytological studies in Oenothera

at King's College. It was found extremely difficult to obtain satisfactory fixation of the chromosomes when the root-tips were collected from germinating seeds. On the other hand, the freshly developed adventitious roots from the hypocotyl of plants grown in sand gave quite satisfactory fixation with Levitsky's fixative (chromic acid 1 % and formalin 10% in

proportion of 1:1). It is well known that fixation images of plant cells

vary according to conditions of the tissue at the time of fixing; this was

especially apparent in the case of Oenothera roots. Levitsky (193I) mentioned that roots collected from germinating seeds grown at low temperatures, such as 15-16? C, showed chromosomes much shorter and thicker, without revealing constrictions, than when they were grown at higher temperatures such as 32? C. Even the frequency of division figures was found to be much less in the former conditions. This observation has been corroborated and it was found further that best fixation can be obtained if the plants are grown in sand and watered once, 2-3 hr. before fixation.

Paraffin sections were made, 8-14ut thick, and the preparations were stained with Feulgen-light green following the method given by Semmens and Bhaduri (I939) and Bhaduri (I938), as well as with Newton's gentian violet iodine method. To obtain clearer preparations and better definition of the microscope image it was found necessary to replace Canada balsam

(which is generally acidic) by another medium which is called "Sira" mountant and is prepared by Messrs Stafford Allen and Sons, Ltd., London. The optimum time of hydrolysis of sections for Feulgen staining was found to be 20 min.

For the study of catenation, observations were chiefly made from smear

preparations, following the method given by Catcheside (I935). In addi-

tion, flower buds were also fixed in Medium Flemming after a pretreatment in Carnoy's fluid and subsequent washing in water.

The observations and drawings were made using a Zeiss 2 mm. apochro- matic objective 1-4 N.A. with achromatic aplanatic condenser 1-3 N.A.,

homogeneous immersion and compensating eyepieces x 20 and x 15.

PREVIOUS WORK ON THE SOMIATIC CHROMOSOMES OF OENOTHERA

The study of the somatic chromosomes of Oenothera began with the observations of Geerts (I907) on O. Lamarckiana. He observed differences in the lengths of the somatic chromosomes. Levitsky (i93 ) later pointed out that "how far these differences are determined by actual chromosome dimensions and how far by the position of the latter with regard to the

optic plane remains unknown". The next observations were made by

355



P. N. Bhaduri

Gates (I912) in a critical study of the nuclear changes during the whole mitotic cycle of 0. lata, a mutant of 0. Lamarckiana with 2n = 15 chromosomes. He suggested that the lata characters are probably constantly associated with the presence of the extra chromosome. He also found some

stages showing a case of "somatic reduction ". Lutz (I916), after examining the chromosomes of 0. Lamarckiana and some of its mutations, came to the conclusion that the somatic chromosomes do not show any difference in dimensions and shape. She also found a small additional chromosome

fragment in some of the mutants. Davis (I909) made a general study of the behaviour of chromosomes during first premeiotic mitosis in the arche-

sporial cells of the anther of 0. grandifora. He found that the somatic nucleus contained chromatic bodies which frequently approximated to the 2n number, i.e. 14. He suggested that they probably represented prochromosomes. In a later paper, Gates and Thomas (I914) found "light areas" in the chromosomes either at the middle or near the end of a chromosome. That most of these light areas correspond to the constriction

region of the chromosome has been discovered quite recently. Hance ( 918) made a detailed study of the somatic chromosomes of 0. scintillans, another trisomic mutation of 0. Lamarckiana, and found the longest chromosome to be nearly 1 8 times the length of the shortest. Levitsky (193 I) concluded that such variation in the dimensions of chromosomes was due to the fact that Hance had made the measurements from drawings, i.e. the projection images of the chromosomes, at the same time disregarding the position of the chromosomes in relation to the optic plane. Van Overeem (I922)

classified into four groups the chromosonmes of 0. Lamarckiana. De Vries and Boedijn (I923, I924), while trying to analyse the chromo-

somes of 0. Lamarckiana and several of its mutations, drew conclusions

indirectly as to their lengths. Levitsky ( 93 ) measured carefully, according to his own devised method, the lengths of the chromosomes and concluded that 0. Lamarckiana shows insufficient length differences. The dimensions

vary gradually, the smallest differing from the largest by 1-35 times.

He, however, found conspicuous differences in the shape of the chromosomes and classified them into four groups. The first consists of three pairs of more or less equal-armed chromosomes, the second of two sharply unequal-armed pairs and the third of one pair of unequal-armed chromo-

somes, but of smaller size. The fourth, a most conspicuous pair with a big appendage at one end of an arm separated by an achromatic zone. Dar-

lington (I93 ) published drawings of the somatic chromosomes of a few

species and hybrids of Oenothera. He found an unusual complexity of the visible structure of the chromosomes. They could not be paired. He

356




suggested that the complicated behaviour of the chromosomes was due to the extreme heterozygosity of the species. Catcheside (I932), while com-

paring the somatic chromosomes of a haploid 0. blandina and the normal

diploid, found that there is a clear correspondence of chromosome types in the two. Marquardt (1938) has made some critical observations regarding the morphology of the somatic chromosomes of 0. Hookeri. He, however, based his observations on nuclei undergoing mitotic divisions in anther tissue.

OBSERVATIONS

The diploid number in all the species and hybrids examined is fourteen, the stable number of the genus. Exceptionally, however, in some root-tip cells of 0. blandina, fifteen chromosomes have been clearly counted (figure 4). The presence of this extra chromosome has been ascertained to be due to real fragmentation and is not an artefact. Though a large number of metaphase plates have been examined for each species, in no other case has fragmentation of chromosomes been observed. It appears further that this fragment belongs to one of the long chromosomes to which it is normally attached by a secondary constriction (figures 3 and 17).

Though the chromosomes of Oenothera species are very short (3-8-2-5,1), a marked difference in length between the longest and the shortest chromosomes, as will appear from table 1, has been observed for each species and hybrid.

TABLE 1 Measurements of

longest and shortest Name of the species and hybrids chromosomes in It

O. Hazelae var. parviflora 3-5-2-5 0. angustissima var. quebecensis 3-6-2-6 Oenothera St Jerome 3-6- 25 Oenothera sp. from Saskatchewan 3-8-2-5 O. Lamarckiana 3-8-2-5 0. blandina 3-8-2-5 0. biformiflora var. cruciata x O. Lamarckiana 3.8-2-6

De Vries and Boedijn (1923) found the range of chromosome length to be 15 to 6 units in 0. Lamarckiana. Hance (I918) calculated it for 0. scintillans as varying from 4-7 to 2-6 units. Darlington (i93i) found the limits from 2-5 to 1-8/, to be general for Oenothera species. According to Levitsky (1931) they are between 2-7 and 20/,u in O. Lamarckiana. Catche- side (I932), on the other hand, finds them to be between 3-6 and 2.8/c in 0. blandina. It will be seen from table 1 that the measurements correspond fairly to those observed by Catcheside. Whatever be the cause of

357



358 P. N. Bhaduri

LFLSMsL~ LH

MFI jlI

M M;0 M 3 4 4

1-16. aphase plates from different species and hybrids of Oenother

var. quebeccrsis, note the LS chromosome with the big appendage. 6. O. Hoo8eri,

SM 56 7

two pairs with secondary constrictions. Note that soe other

lJK MM MMy S

Jer9 10 1

13)M? "

10. L ^

3-4. 0. bldina, note 15 chromosomes including the fragment c in figure 4. The

14-2. O. bifor0iora var. crucaa x O. Lamarckkiana, note the heteromorphic pair of chromosomes L- and LS. In figure 2, only three romosome with secondary constriction. 16. Somatdistinguisha; note the

fragment probably belongs to the chromosome lying left of itio. 5. 00. anustissima var. quebecensis, note the LS chromosome with the big appendage. 6. 0. Hookeri, two pairs of chromosomes with secondary constrictions. Note that some other chromosomes have terminal granules. 7-8. 0. ammophiloides var. laurensis. 9. 0. Hazelae var. parvifora. 10. Oenothera sp. from Saskatchewan. 11. Oenothera St Jerome. 12. 0. biformifora var. cruciata. 13. 0. Hazelae (from Lockeport, N.S.). 14. 0. biformifora var. cruciata x 0. Lamarckciana. 15. 0. missouriensis. Sc = chromosome with secondary constriction. 16. Somatic anaphase in 0. blandina; note the

long pair of chromosomes with marked secondary constrictions, x 2800.




this anomaly in the measurements of different authors, it can be safely concluded from the present observations that the shortest chromosomes differ appreciably in length from the longest ones and this difference is

fairly constant in all the species and hybrids examined. There is a distinct gradation in sizes of the chromosomes in Oenothera.

The intermediate-sized chromosomes between the two extremes merge on either side, sometimes making their separation as a distinct group im-

practicable. This can be verified from table 2.

TABLE 2. SHOWING THE MEASUREMENTS 01E ALL THE

FOURTEEN CHROMOSOMES IN SOME SPECIES

Measurements in mm. of individual chromosomes taken from camera lucida drawings

A-~~~~~~_. Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14

O. Lamarckiana 18-5 18.5 17-5 17-5 15 15 14 13 13 13 12 12 10 10 0. blandina 18 18 17 16 15 15 13 13 13 13 12 11 12 10 O. Hazelae 18 18 16 16 1515 1414 13 13 12.5 12-5 11 11

The primary constriction in a chromosome of Oenothera is less con-

spicuous than the secondary constriction. The latter is remarkably big and the shorter arm of the chromosome (the appendage) separated by a

secondary constriction sometimes gives the false appearance of a chromosome fragment. Unlike other species and hybrids, the secondary constrictions in one pair of chromosomes of 0. Hookeri are very short (figures 6, 20). Besides the primary and secondary constrictions, some chromosomes often show other structural peculiarities. There are sometimes well differentiated globular bodies at either end of a chromosome (figures 6, 13, 15). This is especially clear in preparations stained with gentian violet. These bodies correspond with the terminal granules frequently observed at the ends of chromosomes in diakinesis (figures 64, 65, 74). The very early separation of the chromatids, especially at the ends of a chromosome, often

gives the false appearance of a split trabant (figures 8, 10, 13). This pecu- liarity is commonly met with during the late prophase stages as well as in metaphase chromosomes. Besides the aforesaid peculiarities, one or more light areas in the body of the chromosome (especially with gentian violet staining) sometimes gives a misleading appearance of minute constrictions. The last two kinds of peculiarities are, however, not constant. Such complicated appearances of chromosomes have given rise to con-

flicting ideas regarding the interpretation of the morphology of the somatic chromosomes in Oenothera.

Vol 128. B.

359

24



360 P. N. Bhaduri

The marked secondary constrictions consist of two filaments representing the continuation of the chromatids (figures 30, 39). The appendage which is fixed to the arm of a chromosome by a long filament is, in most of the species, fairly big. The filament gives a positive Feulgen reaction in the same manner as the body of the chromosome. The general appearance

Lvfl 17

18

19 20

21 22

iI>) j 23 24

25

FIGURES 17-25. Showing the comparative morphology of the chromosomes with

secondary constrictions in different species and hybrids of Oenothera. 17. 0. blandina, note the two homomorphic pairs. 18. 0. Lamarckiana, note the heteromorphic pair of long chromosomes. 19. 0. angustissima var. quebecensis; one of the five chromosomes has a very big appendage and a long secondary constriction. 20. 0. Hookeri. 21. 0. ammophiloides var. laurensis; first pair seems to be heteromorphic. 22. 0. Hazelae var. parviflora. 23. Oenothera sp. from Saskatchewan. 24. Oenothera St Jerome. 25. 0. biformifiora var. cruciata x O. Lamarckiana. x 2800.

of the secondary constriction varies considerably. Sometimes in the same section one finds, for instance, the length of the filament varying just as the length of the chromosome does. This is obviously due to shrinkage or swelling effect produced by the fixatives. After examining the chromosomes in different species of Oenothera, it becomes impossible to maintain a sharp distinction between a chromosome with secondary constriction and a satellited chromosome (figures 17-25).

After careful examination of a large number of plates and after using




Feulgen-light green staining it was found that the chromosomes in each

species can be classified into groups according to their shapes and sizes and the position of the primary and secondary constrictions. The following groups of chromosomes have thus been identified; long, medium and short, with median or submedian primary constrictions and chromosomes

possessing secondary constrictions. The general idiogram determined for each species and hybrid as well as the morphology of the chromosomes with secondary constrictions is shown in table 3.

It will be observed from table 3 as well as from table 4 that there are four chromosomes with secondary constrictions in all the species and hybrids examined, except in 0. angustissima var. quebecensis where the number is five. These four chromosomes can be paired into two types in most of the

species (figures 17-25). Not infrequently the two chromosomes appearing as a homologous pair differ appreciably in the respective lengths of their arms (cf. table 2). Such differences are in some species fairly constant, especially in chromosomes with secondary constrictions (figures 17, 22, 23) and lead to the view that corresponding chromosomes in the two complexes of a species may differ in length. In 0. Lamarckiana the two long chromosomes forming a pair are distinctly heteromorphic (figures 2, 18). In 0. ammophiloides the four chromosomes look alike at first but it can be seen that three have submedian primary constrictions and one a median. It appears that this species has also a heteromorphic pair of chromosomes

(figures 7, 8 and 21). In 0. angustissima var. quebecensis again, besides the two pairs of chromosomes, the fifth one, LS, has a big appendage separated by an unusually big secondary constriction (figures 5, 19). In 0. blandina and 0. Hookeri the four chromosomes form two distinct pairs (figures 17, 20) but in the former species one chromosome appears distinctly shorter than its partner. The appendages in this pair of chromosomes also look like trabants. These two pairs of chromosomes correspond to two pairs of nucleoli present in the somatic nuclei of 0. blandina which remain attached to the fused nucleolus during somatic prophase (figure 28). It seems therefore probable that the supernumerary constrictions observed by Catcheside (1932) in this plant do not represent true secondary constrictions. In 0. Hookeri one pair of chromosomes have remarkably big secon-

dary constrictions whereas in the other pair they are hardly distinguishable (figures 6, 20). These four chromosomes can be identified precisely from the rest during prophase, where they only remain attached to the fused nucleolus by their secondary constrictions (figure 30).

It will be observed from table 4 that the maximum number of nucleoli in the root-tip nuclei corresponds to the number of chromosomes with

24-2

361



Table 3. SHOWING THE MORPHOLOGY OF THE CHROMOSOMES IN SOME OF THE DIFFERENT

SPECIES AND HYBRIDS OF OENOTHERA

Species 0. Hazelae var. parviflora

0. angustissima var. quebecensis

Oenothera Moose Jaw, Sask.

Oenothera St Jerome

0. Lamarckiana

0. blandina

0. biformiflora var. cruciata x 0. Lamarcciana

General idiogram 2LM + 2LS + 4MM + 4MS + 2SM

4LM + 1LS + 3MM + 4MS + 2SM

6LM + 4MM + 2MS + 2SS

4ML + 6MM + 2MS + 2SM

3LM + 1LS + 6MM + 2MS + 2SM

2LS + 2LM + 8MM + 2SM

2LS - 4MM + 4MM S + 2SM + 2SS

Morphology of chromosomes with secondary constrictions

2LS and 2MS. One MS chromosome longer than its partner

ILS and 4MS. The appendage and constriction in the LS chromosome are very big

2MS and 2SS. One SS chromosome longer than its partner

2LM and 2SM

1LM, 1LS and 2MS. The secondary constriction in one of the MS chromosomes more marked than in its partner

2LS and 2SM. The appendages of the SM chromosomes appear like satellites

2MS and 2SS. The appendages of SS chromosomes look like satellites

Figures 9, 13, 22

5, 19

10, 23

11, 24 t

1, 2, 18

3, 17

14, 25

LM Long chromosome with median primary constriction. MM Medium chromosome with median primary constriction. SM Short chromosome with median primary constriction.

LS Long chromosome with submedian primary constriction. MS Medium chromosome with submedian primary constriction. SS Short chromosome with submedian primary constriction.



TABLE 4. SHOWING THE RELATIONSHIP OF THE CHROMOSOMES TO NUCLEOLI IN DIFFERENT

SPECIES AND HYBRIDS OF OENOTHERA

No. Species 1 O. Hazelae var. parviflora, 101

from Port George, N.S.

2 0. Hazelae, from Lockeport, 111( N.S.

3 Oenothera St Jerome, Quebec 117

4 Oenothera, sp., from Moose Jaw, 94 Sask.

5 0. ammophiloides var. 138 laurensis

6 0. biformiflora var. cruciata 141

7 0. biformiflora var. cruciata x 204, Typ 0. Lamarckiana 204, Typ

8 0. Lamarckiana

9 0. blandina

10 0. Hookeri

11 0. missouriensis 169/3!

12 0. angustissima var. quebecensis 136

No. of chromosomes

Culture with no. Maximum secondary

(1938) catenation constrictions (14) 4

a (14) 4

(14) 4

(14) 4

(14) 4

(14) 4

)eI (10), (4) 4 ?e II (8), (4)

(12) 1II 4

7nI 4

7ni 4

9 7II 4

(14) 5

Maximum no. of

nucleoli Size differences in somatic of nucleoli in

nuclei somatic nuclei 4 1 big, 1 small,

2 intermediate 4 1 big, 1 small,





2 intermediate 4 1 big, 1 small


2 intermediate 4 2 pairs, one pair

smaller than the other

4 2 pairs, one pair smaller than the other

4 1 big, 1 small, 2 intermediate

5 All different

No. of chromosomes attached to nucleolus

during somatic prophase

4

4

4

4

4

4

4

4

4

4

4

5

No. of nucleoli and their

sizes during diakinesis

1 big, 1 small

1 big, 1 small

1 big, 1 small

2 big, 1 small

1 big, 1 small

1 big, 1 small

1 big, 1 small

1 big, 1 small

Figures 13,22,42,43, 44, 49

9,50

12, 24, 35, 37, 55*

10,23,34,53, 54*

7, 8, 21, 38 to 41, 57, 58

12, 48*

14,25,46,47, 59 to 63*

1, 2, 18, 26, 27

3, 17, 28, 29

6, 20, 30, 31

15,45

-5, 19, 32, 33

* Catenation determined for the first time.

<-1 c

c~ I-

CD

OD

I



P. N. Bhaduri

secondary constrictions; this number being four in all the species and

hybrids examined except in the case of 0. angustissima var. quebecensis where the number is five. It is very difficult to determine this maximum number of nucleoli in small nuclei like those of Oenothera, because they generally fuse in early telophase and the number which is seen in later stages does not

represent the maximum number for the species. Besides, in gentian violet

preparations the small chromatic bodies representing the heterochromatic

portions of chromosomes take up the stain similarly to nucleoli. During early stages, when the nucleoli are still very small, it becomes very difficult to distinguish between the two, thereby making the observation with this stain quite unreliable. With Feulgen-light green stain, however, the maximum number could be determined with fair accuracy during the early telophase when the young nucleoli lie separate from each other.

A distinct and constant size variation of the nucleoli is present in each

species and hybrid. It will be noticed from table 4 and figures 27, 29, 31, 33, 37, 40, 41, 45, 47 and 48, that in all the species and hybrids examined, except 0. Hookeri and 0. blandina, there are one big, one small and two intermediate sized nucleoli. In 0. Hookeri, there is one big pair and one

very small pair of nucleoli, corresponding to the two different pairs of chromosomes with secondary constrictions (figures 6, 20, 31). In 0. blandina also, there are two sizes of nucleoli corresponding to two pairs of chromosomes with secondary constrictions (figures 3, 17, 29).

Although it was not possible to trace the origin of the four nucleoli in reference to particular portions of chromosomes in every case, their origin at the secondary constrictions could easily be inferred from the study of the nature of attachment of particular chromosomes to the nucleoli during prophase. In figure 43 it will be seen clearly that one small nucleolus, lying in the region of secondary constriction of a chromosome, just fitted in as if indicating its point of origin. It will be observed further from table 4 that the number of chromosomes attached to the nucleoli corresponds to the maximum number of nucleoli and the number of chromosomes with

secondary constrictions present in that particular species. As the chromosomes with secondary constrictions alone remain attached to the fused nucleolus during prophase they can be identified precisely from the rest in respect to the metaphase chromosomes (figures 28, 30, 32, 34, 35, 42 and 46). As we know that nucleoli are organized only at secondary constrictions, a count of the number of chromosomes attached to the nucleolus is always an important guide for establishing the maximum number of nucleoli in a species.

364




26 27 8 29 @ (A 28 29

32 33 34

38

Yi .----

'MS 43

47

31

36

365

35

40 41

( .

44 5

45

48

46 FIGURES 26-48. Showing attachment of chromosomes to the fused nucleolus during prophase and number and sizes of nucleoli during telophase in somatic nuclei of different species and hybrids of Oenothera. 26-27. 0. Lamarckiana. 26. Four chromosomes attached to the nucleolus and the remaining ten lying free. 27. One big, one small and two intermediate nucleoli. 28-29. 0. blandina. 28. Four chromosomes attached and the remaining ten lying free. 29. Two pairs of nucleoli. 30-31. 0. Hookeri. 30. The two pairs of chromosomes attached. 31. Two pairs of nucleoli. 32-33. 0. angustissima var. quebecensis. 32. The five chromosomes attached and the remaining nine lying free. 33. Five nucleoli of different sizes. 34. Oenothera sp. from Saskatchewan. 35-37. Oenothera St Jerome. Note in figure 36, one chromosome showing the long filament on the surface of the nucleolus. 38-41. 0. ammophiloides var. laurensis. In figure 39, the secondary constriction of the chromosome attached to the nucleolus appears to consist of two filaments. 42-44. 0. Hazelae. In figure 43, the small nucleolus at the secondary constriction can still be distinguished from the other fused nucleolus. n =nucleolus; MS = medium chromosome with submedian primary constriction. LS = long chromosome with submedian primary constriction. 45. 0. missouriensis. 46-47. 0. biformiflora var. cruciata x O. Lamarckiana. 48. 0. biformifora var. cruciata. x 2000.

37

42

\



P. N. Bhaduri

A definite relationship between the sizes of the nucleoli on the one hand and the dimensions of the appendages or the lengths of filament on the other, could not be established during the present study, although variation in all these characters is very conspicuous in most of the species. Lesley (1938), in tomato, has found that an increase in the dimensions of the satellite is accompanied by an increase in the dimensions of the nucleolus. It seems very likely that the size of the nucleolus is dependent upon the specific capacity of the chromosomes to produce a definite amount of nucleolar substance and not merely on the length of the filament or dimensions of the satellite.

Due to the marked condensation of the chromosomes during diakinesis and the general swelling caused by the fixatives, the secondary constrictions of chromosomes become obscure at this stage. The tiny globular bodies at the ends of the chromosomes, the terminal granules, often give the mis-

leading appearance of trabants but are much smaller than the big appendages and are not separated by the marked secondary constrictions so characteristic of the somatic chromosomes of Oenothera. Clear cases showing secondary constrictions as well as the attachment of the chromosomes to the nucleoli have been observed in a few cases (figures 52, 56, 68, 70, 74). Although four chromosomes with secondary constrictions cannot be identified during diakinesis, indirect evidence shows that the conditions are similar in the pollen mother cells to those in the somatic nuclei. From table 4 it will be seen that the number of nucleoli during diakinesis does not correspond to the maximum number four. This is due to fusion of nucleoli prior to diakinesis. In most of the cases two nucleoli, one big and one small were detectable (figures 52, 56, 58, 60, 63, 71, 72), although in some, three nucleoli of different sizes were observed (figures 54, 78). The

presence of two nucleolar bodies in one nucleolus (figures 79) also con- firms the view that the particular nucleolus is a fusion product of at least two separate nucleoli. It appears therefore that the two nucleoli com-

monly seen during diakinesis really represent the fusion of four nucleoli

corresponding to the four chromosomes with secondary constrictions. A nucleolus takes up a particular position in reference to the ring of

chromosomes. In O. Hazelae for instance one small chromosome with a split satellite always remains attached to a small nucleolus (figure 52). A systematic study of the position of nucleoli with reference to the catenations could not be made during the course of this investigation, as the catenation study was chiefly made from smear preparations, stained with gentian violet, in which the nucleoli are not properly stained. Another difficulty encountered was the frequent appearance of the nucleoli lying

366




'\M X t 49 51

54 55

56

59 6061 62

FIGURES 49-67. 49-52. 0. Hazelae. 49 and 50. Showing (14) in two different strains. 51. Two nucleoli, one big and one small at different positions in the broken ring. 52. One small chromosome attached to the small nucleolus by a split satellite. 53-54. Oenothera sp. from Saskatchewan. 53. (14). 54. Three nucleoli of different sizes, two attached and one free. 55-56. Oenothera St Jerome. 55. (14). 56. One big and one small nucleolus: chromosomes with secondary constrictions (Sc) attached to two nucleoli. 57-58. 0. ammophiloides var. laurensis. 57. (14). 58. One big and one small nucleolus; note the change in the shape of the big nucleolus. 59-63. 0. biformifora var. cruciata x O. Lamarckiana. 59. Type II, showing (8), (4), II. The free pair is attached to the nucleolus. 60. Type II; the small nucleolus appears to be attached to the chain of four, the other nucleolus lying free. 61. Type I; nucleolus attached to chain of four, the dark body on the nucleolus is the nucleolar body. 62. Type I, (10), (4); the fused nucleolus attached to (10). 63. Two pairs of chromosomes attached to nucleoli, one big and one small. 64. 0. biformifora x O. Lamarc- kiana, showing (10), 2II; t terminal granule. Nucleolus attached to (10). 65. Oenothera St Jerome x O. Lamarckiana, showing (10), 211. 66. 0. deflexa var. bracteata x O. Lamarckiana showing (6), (4), 2II. 67. 0. deflexa x O. Lamarckiana showing (10), 2Ii. x 2000.

(

367

5253

58

63

66



P. N. Bhaduri

appressed to the nuclear membrane and quite apart from the chromosomes

(figures 60, 67, 78). This appearance, however, is due to fixation effects, which can be verified by examination of a large number of nuclei.

The catenation was determined in a number of species and hybrids. The results are presented in table 5.

A single trisomic mutation of O. Hazelae var. parvifora, arose in one culture in 1938. The plant was dwarf with markedly narrower leaves and flowers smaller than the parent type. Several flowers were selfed but none set seeds. The chromosome number was found to be fifteen (figures 75, 77, 80). During meiotic prophase in the pollen mother cells of this mutant two nucleoli, one big and one small, have been found to be a general condition, as in the parent. In exceptional cases, however, three nucleoli were observed (figure 78). There is generally a small spherical, deeply stained body on the surface of the nucleolus, representing the nucleolar

body or endonucleolus. Sometimes two such bodies are present in one big nucleolus (figure 79). Irregular distribution of the univalents during first meiotic anaphase has been observed in a few cases, but the general condition is an eight and seven distribution to the two poles (figure 80). The catenation of chromosomes during diakinesis is very irregular. The maximum is a chain of fifteen, though catenations showing 14 + 1, 11 + 2I,; 11 +4; etc., have been observed (figures 75, 76, 77). Non-disjunction in the

pollen mother cells leading to eight-and-six distributions has been observed

quite frequently in the parent O. Hazelae (figure 81). In one batch of root-tips of O. Hazelae the chromosomes at metaphase

appeared to show somatic pairing. Sometimes each pair was found to remain attached at one end (figure 82). It was found subsequently from well fixed material that this pairing was only chance association brought about by bad fixation. Marquardt (I938) has observed similarly in the anther archesporium of O. Hookeri, cases of apparent pairing of somatic chromosomes. He concluded that the mutual co-ordination between chromosomes was not so marked as to be described as regular somatic

pairing. His suggestion that this association of the chromosomes may represent a step towards conjugation, which will take place in the following nuclear division, does not appear to be sound from the present observation.

Distinct chromatic bodies (prochromosomes) scattered about in the

resting nuclei of root-tip cells have been observed in all species of Oenothera. The number of these bodies corresponds to the number of chromosomes

(figure 83) and they are Feulgen positive. Some of them, generally four, remain in close association with the nucleolus. Doutreligne (1933) showed that these bodies are particular portions of chromosomes representing the

368




TABLE 5. CATENATIONS OF SOME INTERSPECIFIC HYBRIDS

AND ONE TRISOMIC MUTATION OF OENOTHERA

Name of the No. parent species

1 0. biformiflora var. cruciata x 0. Lamnarckiana

2 0. biformiflora var. cruciata x 0. Lamarckiana

3 0. biformifora var. cruciata x 0. Lamarckiana

4 0. Lamarckiana x 0. biformiflora var. cruciata

5 0. biformifora x O. Lamarckiana

6 0. biformifora x 0. Lamarckiana

7 0. Lamarckiana x 0. biformifora

8 O. deflexa x 0. Lamarckiana

9 0. deflexa var. bracteata x 0. Lamarckiana

10 0. pycnocarpa var. parvifora x 0. Lamarckiana

11 0. pycnocarpa var. cleistogama x 0. Lamarckiana

12 0. pycnocarpa var. cleistogama x 0. Lamarckiana

13 Oenothera St Jerome x 0. Lamarckiana

14 0. Hazelae (trisomic mutation)

Culture no.

(1938) 204*

Principal distinguishing characters of the segregate

types in F1 Type It

204.1.9* Type It

Maximum catenation

(10) +(4)

(10)+ (4)

204 Type IIT (8) +(4)+ 1I

205* Type IIt (8)4-(4) + 1

206 Type It (10)+2II

206 Type II. (10) +21i

207 Type II- (8)+(4) + 1

213 Type II$ (6)+(4)+ 21

214 Type II$ (10)+ 21

222 Type IIt (10)+ 211

224 Type It (10)+(4)

Figure no.

61, 62

59

64

66

67

73

68

224 Type II+ (8)- (4)+1lI

209 Type III

102.1.1 15; 14+ 1; 13 + 1

65

75 to 77

* All the plants belonging to cultures 204 and 205 had cruciate flowers except one plant (204.1.9) which produced flowers with three types of petals, broad, cruciate and intermediate.

t Type I = leaves with white midrib. $ Type II = leaves with pink midrib.

369



370 P. N. Bhaduri

68 69 71 70

72 ^ "

72 73 ---74 75

76

O'.

80 81 82 83

FIGURES 68-83. 68-72. 0. pycnocarpa var. cleistogama x 0. Lamarckiana. 68. Type I, showing (10), (4), one chromosome attached to the nucleolus by a secondary constriction. 70. Type II; (8), (4), 1I. Two chromosomes with secondary constrictions placed end to end in the chain of four. 71. Type I, one big and one small nucleolus fusing. 72. Type I; the small nucleolus attached to chain of four and the big one to (10). 73. 0. pycnocarpa var. parvifora x 0. Lamarckiana, showing (10), 2II; the nucleolus attached to (10). 74. (10), (4), in 0. pycnocarpa var. cleistogama x 0. Lamarckiana, Type I; the (4) is broken. 75-80. Trisomic mutation of 0. Hazelae. 75. 11+2I. 76. Chain of 15. 77. 14+ 1. 78. Three nucleoli; e= nucleolar body, v= vacuole. 79. Zygotene; one nucleolus with two nucleolar bodies. 80. 7 and 8 distribution during anaphase I. 81. Metaphase II in pollen mother cells of 0. Hazelae showing 8 and 6 grouping due to non-disjunction. 82. False somatic pairing. 83. Prochromosomes, stained with Feulgen; four of them in close association with the nucleolus. Root-tip cell of Oenothera sp. from Saskatchewan. Figures 68-81, x 2000; figures 82-83, x 2800.




regions containing the spindle attachment constrictions. The presence of distinct chromatic bodies in the resting nuclei of somatic cells was observed

long ago by Davis (I909) in 0. grandiflora and by Gates (I912) in 0. lata.

Marquardt (1938) has shown in O. Hookeri that these bodies are the heterochromatic middle portions of chromosomes, as compared to the euchromatic end segments, which following condensation give the typical appearance of chromocentres during resting stage.

DISCUSSION AND GENERAL CONCLUSIONS

Fragmentation of somatic chromosomes in Oenothera has until now been

reported only in some of the mutations of 0. Lamarckiana (Lutz, I916; Hance I918; Van Overeem I922). It is now realized that part of the observation regarding fragmentation by previous authors was due to

misinterpretation of the remarkably big secondary constrictions so characteristic of Oenothera. It is quite certain, however, that there must be real fragmentation as well to account, for instance, for the observations of Hance (I918) who counted 21 chromosomes in some of the somatic cells of 0. scintillans. Taking into consideration that fragmentation has not been observed in any other species or hybrids but in 0. blandina and the

previous observations of fragmentation in some mutations of O. Lamarc-

kiana, it appears very likely that this phenomenon expresses a peculiar condition of certain chromosomes in mutations of O. Lamarckiana, probably indicating weak points in those chromosomes where they can break.

It has been shown already that in most species and hybrids of Oenothera, there are four chromosomes with secondary constrictions corresponding to four nucleoli in the root-tip cells. The extraordinary situation in 0. angustissima var. quebecensis, where there are five secondary constrictions and five nucleoli, can be explained by the fact that this species shows certain

morphological characters quite distinct from other Onagrads. In fact, as

suggested by Gates and Catcheside (I932) and Gates (i933), this species shows characters suggesting an intermediate position between the two

subgenera, Onagra and Raimannia. Very little information is available

regarding the somatic chromosomes of any species belonging to the sub-

genus Raimannia. One can only make out from the figures given by Darlington (I93I) for 0. Berteriana that there are more than two, maybe four to seven, chromosomes with secondary constrictions. In the absence of cytological information on species of Raimannia, nothing can be said as to the origin of 0. angustissima var. quebecensis. It will be very inter-

371



372 P. N. Bhaduri

esting to know in this connexion the conditions in such species as 0. cam-

pylocalyx Koch and Bouche, which show distinct Raimannia characters. It has been shown definitely that in 0. Hookeri, like other species, there

are two pairs of chromosomes with secondary constrictions corresponding to two pairs of nucleoli. Marquardt (I938), on the other hand, claims that there is only one pair of Sat-chromosomes in 0. Hookeri. He has criticized the observations of Wisniewska (I935), who found three more pairs of chromosomes with a round appendage at the end of one arm, and he

explains that such appendages are not a constant natural feature of a

particular chromosome. He further claims that his conclusion is correct on the ground that he failed to observe such appendages in preparations stained with aceto-carmine, the latter being the classical method for

distinguishing heterochromatin from euchromatin. It seems that Mar-

quardt has failed to see the other pair of chromosomes with secondary constriction due to the fact that he based his observations on anther

tissue, where it is well known that the fixation of the chromosomes is

definitely poorer. He missed the pair of chromosomes with very short

secondary constriction. In support of this conclusion, the observations of Catcheside (I932) can be cited. He found that the chromosomes in the anther tissue are much shorter and thicker than those in the root-tips. The problem of fixation in Oenothera has already been discussed.

It is interesting to learn that while a pair of chromosomes with secondary constrictions are heteromorphic in 0. Lamarckiana, in 0. blandina, a

homozygous mutation of O. Lamarckiana without catenation, these chromosomes are homomorphic. One concludes that structural changes of chromosomes in 0. blandina (probably due to segmental interchange) have not only brought back homozygosity in the species but also the morphological homology of a certain pair of chromosomes. The view that the

heteromorphic nature of the Sat-chromosomes indicates heterozygosity of the individual seems to have obtained a direct proof from the evidence

just put forward. It is interesting further to note that in O. Lamarckiana the nucleoli

are of different sizes and cannot be paired, while in 0. blandina the nucleoli can be paired into two sizes corresponding to two pairs of chromosomes with secondary constrictions. Presence of unpaired nucleoli therefore indicates heterozygosity of the species. This is further substantiated by the fact that in O. Hookeri, a homozygous species without catenation, there are also two distinct pairs of nucleoli corresponding to two pairs of chromosomes with secondary constrictions. The rather extraordinary situation in 0. missouriensis (without catenation) where the nucleoli could




not be paired, cannot be explained at present till further cytological information is available. It must be pointed out here, however, that the roots from which the present observations are made, were collected from seeds produced by crossing two strains of 0. missouriensis. The female

parent shows no catenation (Hedayetullah i933) and is completely self- sterile (Gates I939).

The genetic significance of the number of nucleoli in plant tissue was first realized by De Mol (1926) who found that the number of nucleoli in

Hyacinthus may be used as a guide to the number of chromosome sets. Kuhn (I928), after examining thirty-seven species of Thalictrum, found that the species have trabants. He concluded that univalent idiograms apparently have never more than one pair of trabants. These expectations took a positive aspect and harmonized into a general hypothesis since the

discovery by Heitz (I93Ia) of the relation of Sat-chromosomes to the nucleolus. Heitz concluded that the nucleoli as a rule arise in telophase from the satellites in a particular chromosome pair. In a later paper (I93 b), after examining thirty-three species of Vicia, he concluded that all plants probably have satellited chromosomes which give rise to nucleoli in telophase. The functional correspondence of the secondary constrictions to the satellites, in regard to the organization of the nucleoli, has been realized quite recently. In fact, a distinction between the two seems to be rather arbitrary than real. However, the presence of secondary constrictions which do not produce nucleoli has been recorded by a number of workers. Sato (I936, 1938) has found such a condition in Scilla, Zephyr- anthes, Cyrtanthus, Hebranthus and Narcissus, Sato (I937) and Resende

(I937) in Aloinae, and Fernandes (1936) in Narcissus. Until more direct and exact information is forthcoming regarding the nature of the constrictions and their evolution in chromosome-segments, it seems useful to call all constrictions which are related to nucleolar organization "nucleolar constrictions ".

McClintock (I93I) independently came to the same conclusion as Heitz

regarding the relation of Sat-chromosomes and the organization of the nucleoli in the case of maize. She concluded in a later paper (I934) that the number of nucleoli in somatic telophase is correlated with the number of Sat-chromosomes, haploid cells having one, diploids two and triploids three. She has made a very significant observation in maize that certain

genomic deficiencies may lead to the formation of many small scattered nucleoli instead of a fixed number.

Quite an amount of literature has been accumulated on the subject since the discovery of the relation of Sat-chromosomes to nucleoli. The

373



P. N. Bhaduri

problem has been discussed by Gates (I937, 1938) quite recently from several aspects and need not be repeated here. Many observations, con-

tradictory to the general principle, however, have been recorded quite recently, making the relation of chromosomes to nucleoli more com-

plicated than was originally supposed. According to Bushnell (1936) in Labiatae and Okuno (I937) in Lobelia, there is sometimes no relation between the satellited chromosomes and the nucleoli. It appears that some such conflicting observations may be explained as partly due to inadequate technique and to the presence of extremely small trabants, as discovered in some Trillium species (Mensinkai 1939), Haworthia species and others. A judicious and wider application of the phenomena of amphiplasty as described by Navashin (I927, 1934) may also account for the apparent failure of the general principle of correspondence of the number of nucleoli to Sat-chromosomes in certain cases.

Though conflicting data are being put forward from time to time, the

principle that primary diploids have a single pair of nucleoli seems to be a useful guide in the study of the evolution of the nucleus. In fact, many of these negative observations may ultimately help in establishing the

hypothesis as a biological law, rather than to disprove it. Gates (1938) accordingly has pointed out that "Three complementary lines of observation can now be brought to bear on the phylogeny of the nucleus in cases of primary or secondary polyploidy. When these confirm each other, the evidence of former changes, even in the basic number of chromosomes, is on a firm basis."

The application of the principle in the case of Oenothera has met with considerable difficulty. Parthasarathy (1938) already doubted, without

making any direct observation, that the rule of the correspondence of satellites and nucleoli might not be applicable to definitely known segmental interchanged heterozygotes, such as Oenothera, Aucuba, Antho-

xanthum, etc. Contrary to such expectations, however, a close correspondence between the number of nucleoli and the number of secondary constrictions in Oenothera has been established for each species and hybrid of Oenothera studied. The problem still remains, to explain the presence of four nucleoli in both homozygous and heterozygous species of Oenothera which are regarded as true diploids.

McClintock (I934), in Zea Mays, has shown that a change in the number of nucleoli can be brought about in some of the progenies by X-raying the

parent diploid plants. This she explained as due to breakage at the region of the nucleolar body of chromosome 6 followed by an interchange with chromosome 9, the two resulting chromosomes both having a bit of the

374




nucleolar body. Both the chromosomes are therefore able to organize a nucleolus. Microspores of this plant, instead of possessing one chromosome with nucleolar body, possess two such chromosomes. Explanation of the four nucleoli in Oenothera on this basis seems to be unlikely.

It is now known that the linked condition of the chromosomes has arisen within the genus Oenothera. The subgenus Onagra began with large-flowered, homozygous species, with free chromosome pairs from which were developed species with progressively increasing chromosome catenation. The reverse

process, by which more homozygous forms with no chromosome linkage arise as mutations from heterozygous ancestors, can also take place.

From the observations recorded in this paper it can be safely concluded that the constant presence of four nucleoli corresponding to four secondary constrictions in both large-flowered homozygous species with no chromosome linkage, such as 0. Hookeri, 0. missouriensis and 0. blandina, as well as in the heterozygous species showing high catenation, such as O. Lamarck- iana, 0. Hazelae, 0. biformiflora cruciata and 0. ammophiloides, is a more

primitive character than the linkage of chromosomes. It further shows, from the behaviour of O. Lamarckiana and 0. blandina, that these two conditions are independent of each other in these species and most likely in others. As already pointed out, there is also reason to believe that 0. Berteriana, the only representative of the subgenus Raimannia whose somatic chromosomes have been examined, also possesses not less than four secondary constrictions and probably the same number of nucleoli in its somatic nuclei, the maximum catenation for the species being a ring of fourteen. It can be reasonably concluded that four nucleolar chromosomes is the minimum number in the genus Oenothera.

Applying the principle of correspondence of the number of Sat-chromosomes to the number of nucleoli in relation to polyploidy, the problem turns to whether Oenothera should be looked upon as essentially a diploid or represents an exception to the principle already mentioned. Certain previous observations, mentioned below, along with the present observations give strong evidence in support of the view that seven may be a derived chromosome number. A very significant observation in this connexion was made by Emerson (I929), who found the almost constant presence of a bivalent and in one case a chain of four univalents in pollen mother cells of a haploid 0. franciscana. Similarly, Catcheside (1932) has shown in haploid 0. blandina that out of 1186 pollen mother cells 218 showed the presence of one bivalent and five univalents, five cells showing trivalent chromosomes and one a quadrivalent. He also recorded multivalent formation, such as trivalents and quadrivalents during diakinesis

Vol. 128. B.

375

25



P. N. Bhaduri

in diploid 0. blandina. He has stressed that the trivalents formed from a bivalent and an univalent represent a true multivalent formation and cannot be interpreted as due to interlocking, etc. He holds that the

presence of multivalents in haploid Oenothera proves the presence of

reduplicated segments in non-homologous chromosomes. The evidence put forward in this paper indicates that the haploid number in Oenothera is a

secondary number. Further cytological observations of this kind in

homozygous species of Oenothera and in related genera, will show the exact basic number for the genus.

In conclusion I desire to express my profound gratitude to Professor R. R. Gates, F.R.S., under whose supervision the present work was carried out.

SUMMARY

1. A comparative study of the relation of chromosomes to nucleoli has been made in nine species and one hybrid of Oenothera. The distinction between a satellited chromosome and one with secondary constriction breaks down in Oenothera. Both the constricted region and the appendage are well marked in most of the species. The filament, like the appendage, is

Feulgen-positive. 2. The chromosomes have been classified in some of the species according

to their sizes, the situation of the primary constriction and the presence of

secondary constrictions (table 3). 3. An exact correspondence between the number of secondary con-

strictions and the number of nucleoli has been established in each case. This number is four in all the species except 0. angzstissima var. quebecensis where it is five. Determination of the position of nucleolar chromosomes in the ring is important as a further means of identifying chromosomes in the two complexes of a species.

4. The presence of four nucleoli corresponding to four secondary constrictions in (a) heterozygous species with high chromosome catenation, such as 0. Lamarckiana, O. Hazelae and 0. biformifiora and (b) homozygous species with seven free pairs such as 0. Hookeri and 0. missouriensis, proves that the presence of four nucleolar chromosomes is an older character than chromosome linkage. This supports the view that ring formation in Oenothera has evolved in the genus.

5. That morphological changes of chromosomes can be brought about by segmental interchange is evidenced by the fact that at least one hetero-

376



Cytological studies in Oenothera 377

morphic pair of Sat-chromosomes in 0. Lamarckiana has become homo-

morphic in its mutant 0. blandina. 6. There is variation in the sizes of the nucleoli in species and hybrids

of Oenothera. O. Lamarckiana has one very small, one quite big and two intermediate nucleoli, whereas in the homozygous 0. blandina and 0. Hookeri there are two distinct pairs of nucleoli. An unpaired condition of nucleoli in Oenothera therefore indicates heterozygosity of the species.

7. Prochromosomes, which are Feulgen-positive, have been observed in somatic cells of some species.

8. The catenation of three species and nine interspecific hybrids has been determined (tables 4, 5). In a narrow-leaved trisomic mutation of O. Hazelae the 15 chromosomes showed irregularities in catenation.

9. The presence of four nucleoli corresponding to four secondary con-

strictions, the frequent presence of multivalent chromosomes in the pollen mother cells of haploid and diploid plants, as well as the fact that no common basic number is found in the family Onagraceae, indicates that the haploid number 7 in Oenothera is a derived number. On this assumption Oenothera species cannot, therefore, be looked upon as true diploids.

REFERENCES

Bhaduri, P. N. I938 J.R. Micr. Soc. 58, 120-124. Bushnell, E. P. 1936 Bot. Gaz. 98, 356-362. Catcheside, D. G. I932 Cytologia, Tokyo, 4, 68-113.

- 1935 Genetics, 17, 314-341.

Darlington, C. D. I931 J. Genet. 24, 405-474. Davis, B. M. I909 Ann. Bot. Lond., 23, 551-571. De Vries, H. and Boedijn, K. 1923 Genetics, 8, 233-238.

- - I924 Ber. dtsch. bot. Ges. 42, 174-178. Doutreligne, J. 1933 Cellule, 42, 31-100. Emerson, S. H. 1929 Cellule, 39, 159-165. Fernandes, A. 1936 Bot. Soc. Broteriana, 11 (ii serie), 87-146. Gates, R. R. 1912 Ann. Bot., Lond., 26, 993-1010.

- 1933 J. Linn. Soc. (Bot.), 49, 173-197. - I936 Phil. Trans. B, 226, 239-355. - 1937 Cytologia, Tokyo, Fujii Jub. Vol. pp. 977-986. - 1938 J.R. Micr. Soc. 58, 91-111.

- 939 Nature, Lond., 143, 245. Gates, R. R. and Catcheside, D. G. 1932 J. Genet. 26, 143-178. Gates, R. R. and Thomas, N. 1914 Quart. J. Micr. Sci. 59, 523-571. Geerts, J. M. 1907 Ber. dtsch. bot. Ges. 25, 191-195. Hance, Robert T. 1918 Genetics, 3, 225-275.

Hedayetullah, S. I933 Proc. Roy. Soc. B, 113, 57-70. Heitz, E. 193 a Planta, 12, 775-844.

- x93 b Planta, 15, 495-505.

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378 P. N. Bhaduri

Kuhn, E. 1928 Jb. wiss. Bot. 68, 382-430.

Lesley, M. M. 1938 Genetics, 23, 485-493.

Levitsky, G. A. 193I Bull. Appl. Bot. Select. 27, 19-174. Lutz, A. M. I916 Amer. J. Bot. 3, 502-526.

Marquardt, H. I938 Z. Zellforsch. 27, 139-210. McClintock, B. 193I Bull. Mo. Agric. Exp. Sta. no. 163, 1-30.

- 1934 Z. Zellforsch. 21, 294-328. Mensinkai, S. W. 1939 J.R. Micr. Soc. 59, 82-112. Mol, W. E. de 1926 Cellule, 38, 7-64. Navashin, M. 1927 Z. Zellforsch. 3, 195-233.

- 934 Cytologia, Tokyo, 5, 169-203. Okuno, S. 1937 Cytologia, Tokyo, Fujii Jub. Vol. pp. 896-902. Van Overeem, C. 1922 Bot. Zbl. 39, 1-80.

Parthasarathy, N. 1938 Cytologia, Tokyo, 9, 307-318. Resende, F. 1937 Planta, 26, 757-807. Sato, D. 1936 Cytologia, Tokyo, 7, 521-529.

- I937 Cytologia, Tokyo, Fujii Jub. Vol. pp. 80-95. - 1938 Cytologia, Tokyo, 9, 203-242.

Semmens, C. S. and Bhaduri, P. N. 1939 Stain Tech. 14, 1-5. Wisniewska, E. 1935 Acta Soc. Bot. Polon. 12, 113-164.

On the hardening of the ootheca of Blatta orientalis

BY M. G. M. PRYOR From the Entomology Department, Zoological Laboratory, Cambridge

(Communicated by A. D. Imms, F.R.S.-Received 13 November 1939)

The ootheca of Blatta orientalis was chosen as the object of an investigation into the hardening mechanism of the insect cuticle because the material of which it is composed looks like the hard brown exocuticle of a typical insect. It is, however, secreted by glands, from which comparatively large amounts of the substances concerned can be obtained.

The ootheca is about 8 mm. long, and has the shape of a carpet bag, opening by two flat adpressed lips, which have a regular pattern of ridges and hollows on them. Inside there are about a dozen elongated eggs, arranged in two rows. In the wall of the ootheca are embedded numerous crystals of calcium oxalate (Hallez 1909). The function, if any, of these

crystals is obscure; they may help to harden the wall, or they may simply be excretory products, since Sinety (1901) has recorded crystals of an oxalate from the Malpighian tubules of a phasmid.

378 P. N. Bhaduri

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Lesley, M. M. 1938 Genetics, 23, 485-493.

Levitsky, G. A. 193I Bull. Appl. Bot. Select. 27, 19-174. Lutz, A. M. I916 Amer. J. Bot. 3, 502-526.

Marquardt, H. I938 Z. Zellforsch. 27, 139-210. McClintock, B. 193I Bull. Mo. Agric. Exp. Sta. no. 163, 1-30.

- 1934 Z. Zellforsch. 21, 294-328. Mensinkai, S. W. 1939 J.R. Micr. Soc. 59, 82-112. Mol, W. E. de 1926 Cellule, 38, 7-64. Navashin, M. 1927 Z. Zellforsch. 3, 195-233.

- 934 Cytologia, Tokyo, 5, 169-203. Okuno, S. 1937 Cytologia, Tokyo, Fujii Jub. Vol. pp. 896-902. Van Overeem, C. 1922 Bot. Zbl. 39, 1-80.

Parthasarathy, N. 1938 Cytologia, Tokyo, 9, 307-318. Resende, F. 1937 Planta, 26, 757-807. Sato, D. 1936 Cytologia, Tokyo, 7, 521-529.

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On the hardening of the ootheca of Blatta orientalis

BY M. G. M. PRYOR From the Entomology Department, Zoological Laboratory, Cambridge

(Communicated by A. D. Imms, F.R.S.-Received 13 November 1939)

The ootheca of Blatta orientalis was chosen as the object of an investigation into the hardening mechanism of the insect cuticle because the material of which it is composed looks like the hard brown exocuticle of a typical insect. It is, however, secreted by glands, from which comparatively large amounts of the substances concerned can be obtained.

The ootheca is about 8 mm. long, and has the shape of a carpet bag, opening by two flat adpressed lips, which have a regular pattern of ridges and hollows on them. Inside there are about a dozen elongated eggs, arranged in two rows. In the wall of the ootheca are embedded numerous crystals of calcium oxalate (Hallez 1909). The function, if any, of these

crystals is obscure; they may help to harden the wall, or they may simply be excretory products, since Sinety (1901) has recorded crystals of an oxalate from the Malpighian tubules of a phasmid.



cytological studies in oenothera with special reference to the relation of chromosomes to nucleoli

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