Theor. Appl. Genet. 61, 35-40 (1982)
9 Spfinger-Vedag1982
Studies on Aneuploids of Petunia Part 1: Cytomorphological Identification of Primary Trisomics V.R. Reddi and V. Padmaja Department of Botany, Andhra University, Waltair 530003 (India)
Summary. The seven possible primary trisomics of Petunia (2 n = 14) located in the progenies of triploid, hypertriploid and hypotriploid plants were distinguished from one another and from diploid on the basis of cytological and morphological criteria. They were provisionally named as "Oval", "Semi", "Slender", "Pseudonormal", "Arrow", "Narrow" and "Giant". In three of the trisomics, the extra chromosome was identified for the first time at pachytene stage. Postpachytene studies revealed no precise relationship between the length of extra chromosome and the frequency of multiple association. Key words: P e t u n i a - Primary trisomics - Morphology - Chromosome associations - Chiasma
Materials and Methods Triploids, which formed the main source for producing trisomics, were derived from the crosses of induced autotetraploids of C2 generation with diploids. They were selfed and crossed reciprocally to normal diploids and sowings were made immediately to raise progenies. Several morphological parameters such as plant height, plant type, leaf shape and size, leaf tip, surface and margin, phyllotaxy, petiole and intemode length were taken into consideration, apart from floral and capsule characters, for classifying the trisomics. Flower buds were fixed in 1:4 acetic alcohol and chromosomal counts for the initial detection of trisomy were made from acetocarmine smears showing diakinesis or metaphase ! stages.
Introduction Results Offspring of triploid, hypertriptoid and hypotriploid plants in the white flowered variety of P. axillaris (Lam.) B.S.P., were examined in order to locate the primary trisomics. Cytological screening of 459 plants representing progenies from 3nX 2n; 2 n • 3n and 4 n x 3 n crosses, revealed 179 to be aneuploid. Although the occurrence of trisomics in the genus has been reported earlier (Levan 1937; Sullivan 1947; Smith et al. 1975; Maizonnier 1976) identification of the trisomic chromosome was made only recently by the application of the fluorescence staining technique to enzymatically macerated root tips (Smith et al. 1975). In the present investigation, it was possible to recognize, apparently for the first time, an extra chromosome in three of the primary trisomics at pachytene, based on recognizable landmarks on the individual chromosomes of the diploid complement (Padmaja and Reddi 1981). This paper describes the results of our first attempt and deals with morphological and cytological characteristics of the seven primary trisomics.
Classification into 7 categories on the basis of mature plant characters was made, which corresponded to the basic number. Leaf shape varied in the different classes, facilitating classification to some extent. Even so, 5 of the 7 trisomics were named after leaf shape, and careful examination was made of all the other characters mentioned in order to provide detailed description of individual classes. The trisomics were named provisionally as "Oval" (Triplo-1), "Semi" (Triplo-2), "Slender" (Triplo-3), "Pseudonormal", "Arrow", "Narrow" and "Giant", and were recovered at different frequencies in the triploid progenies. Meiosis in triploid Petunia revealed that the trisomics owe their origin to the irregular distribution of chromosomes at anaphase I (Reddi and Padmaja 1980). Only 3 of the 7 trisomics could be identifiied at pachytene, while the demarcation between 2 others, "Arrow" and "Narrow", was not certain. A brief description of the morphology of the 7 classes is presented below.
0040-5752/82/0061/0035/$ 1.20
36 Trisomic-I-"Oval". The two cotyledonary leaves, as well as those produced later, were "oval" in shape, with obtuse leaf tips (Fig. 1 b). This is in contrast to the lanceolate shape in the diploids with acute leaf tips (Fig. 1 b). Further, they showed comparatively poor growth rate, erect plant type, with variable stem thickness: Leaves were small, length/breadth ratio less than normal and margin entire, thick and somewhat succulent with short petioles. Capsules, was well as the sepals, were short with obtuse tips, like the leaves themselves. Seed set was almost normal.
Theor. Appl. Genet. 61 (1982) diameter (Fig. ld). Leaves were narrowly lanceolate, tip pointed, margin slightly wavy, dark green in colour, with longer petioles, especially on lower leaves. Flowers of normal size, but with longer pedicels, and purple in colour. Sepals narrow, long and pointed like the leaves themselves. Capsules were long, also with pointed tips. "Pseudonormal". Plants of this trisomic class were in no way different from normal diploids (Fig. 1 e), except for the slow growth rate in the seedling stage. The growth rate was better, however, when compared with plants of other trisomic classes.
Trisomic-2-"Semi". Plants with nucleolar chromosome in trisomic condition and were characterised by broad leaves with acute leaf tips (Fig. 1 c) and thus differ from the pointed leaf tips of the diploids and obtuse tips of Trisomic-1. Length/Breadth ratio was less and they resembled tetraploids in this respect, but, differed from the latter in leaf texture. Capsules did not differ from normal diploids, except for the smaller size.
"Arrow" Identified by the "Arrow"-like shape of the leaves which were characteristically arranged. Erect plant type with medium height, developing 2 - 4 lateral branches; leaves - lanceolate, the width of which gradually decreases from base to top resulting in "Arrow" like shape (Fig. 1 f). The dark green coloured leaves showed compact arrangement, pointed upwards.
Trisomic-3-"Slender'" Plants carrying chromosome-3 in trisomic condition, and were characterised by tall, slender, creeping stem bearing lateral branches, mainly at the distal region which remained thin with reduced
"'Narrow" There was close resemblance between this and "slender" trisomic class. However, the differences were that the leaves of"Narrow" were lanceolate and narrow, consistently yellowish green in colour, stem
Fig. 1 a - h . Morphology of diploid and primary trisomic plants, a Diploid; b Triplo-1 "Oval"; c Triplo-2 "Semi"; d Triplo-3 "Slender"; e "Pseudonormal"; f "Arrow"; g "Narrow"; h "Giant"
37
V.R. Reddi and V. Padmaja: Studies on Aneuploids of Petunia. Part 1 Table 1. Chromosome association in 6 primary trisomics at diakinesis and metaphase I Type of association
Trisomic class
III
"Oval . . . .
II
I
Ring Rod 1 -
PMC's
%
PMC's
1 -
1 1 2 1 3 2 2
7 6 5 6 5 5 4 4 4
1 1 3 1 3 1 3
1
1
4
2
1
1.49
-
1 1 -
3 3 2 4
3 3 3 2
3 2 3
2 5 6 -
2.98 7.46 8.95 -
. 29 19 4
-
4 12 4 . 10 2 15 3 3
Semi . . . .
Total
5.97 17.91 5.97 . . 14.92 2.98 22.38 4.47 4.47
67
Slender . . . .
%
12 11
7.22 6.62
34 40 17 -
20.48 24.09 10.24 -
.
-
.
PMC's
%
PMC's
2 7 4 2 15 5 14 8 4
2.66 9.33 5.33 2.66 20.00 6.66 18.66 10.66 5.33
9 13 . 24 2 27 13 .
1
. 17.46 11.44 2.40
166
Pseudonormal . . . . Arrow . . . .
1.33
.
. 8.00 8.00 1.33
6 6 1 75
thin a n d slender, but n o t so thin as to attain c r e e p i n g habit (Fig. 1 g). F l o w e r s w e r e white in colour, with increased corolla d i a m e t e r , sepals also y e l l o w i s h - g r e e n , n a r r o w a n d long. G o o d seed set was o b s e r v e d o n l y in self p o l l i n a t e d flowers.
"Giant" Trisomic. O n l y two plants w e r e f o u n d with interesting p h e n o t y p e s in w h i c h flower size i n c r e a s e d m a r k e d l y (Fig. 1 h). G r o w t h rate was slow, p l a n t type erect, short m a i n stem w i t h no lateral branches, a n d the leaves w e r e n a r r o w l y l a n c e o l a t e a l o n g the entire m a r gin, o f p o i n t e d tips a n d l o n g pedicels, especially in the lowert ones. T h e y w e r e c o m p a c t l y a r r a n g e d with shorter internodes. O n e o f these plants f l o w e r e d early on 58th day a n d the flowers w e r e larger t h a n in the tetraploids. G o o d seed set was o b t a i n e d , after crossing with diploid, but the h y b r i d seed failed to g e r m i n a t e . Both
. 8 5 -
%
8.91 12.87 . . 23.76 1.98 26.73 12.87 . . . . 7.92 4.95 -
101
PMC's 1 2 3 . 26 2 22 14 3 .
%
PMC's
1.19 2.38 3.57 .
Narrow" %
1 3
1.07 3.22
11 1 33 17 -
11.82 1.07 35.48 18.27
3 9 8 7
3.22 9.67 8.60 7.52
. 30.95 2.38 26.19 16.66 3.57
.
6 4 1
-
. 7.14 4.76 1.19
84
93
plants d i e d for u n k n o w n reasons 2 0 - 2 5 days after flowering, so that n e i t h e r meiosis n o r b r e e d i n g b e h a v i o u r could be studied.
Meiosis in Primary Trisomics. C h r o m o s o m e associations w e r e m a i n l y o f Iil I + 6 . (Fig. 2 e) or 7 . + Ii (Fig. 2 d) as expected. But, in a few cells, u n i v a l e n t s w e r e f o u n d r e p l a c i n g o n e or two bivalents ( T a b l e 1). In a d d i tion, the relative f r e q u e n c y o f ring a n d r o d type o f bivalents, w h i c h t e n d to alter c h i a s m a frequencies, was f o u n d to vary in different trisomic classes ( T a b l e 2). In trisomic-I, d e s i g n a t e d as " O v a l " , the extra chrom o s o m e was p r e s e n t as a u n i v a l e n t in 67.14% o f the cells e x a m i n e d in two plants, while in the rest it f o r m e d a trivalent c o n f i g u r a t i o n ( T a b l e 1). T h e f r e q u e n c i e s o f trivalents a n d u n i v a l e n t s v a r i e d a p p r e c i a b l y in different plants o f the s a m e trisomic class, b u t the a v e r a g e
Table 2. Comparison of frequencies of trivalents, bivalents and univalents in 6 primary trisomics at diakinesis Trisomic class
Diploid control "Oval" "Semi" "Slender" "Pseudonormal" "Arrow" "Narrow"
No. of cells analysed
98 56 166 77 101 61 93
* Significant at 1% level
Average number per cell Trivalents
0.303 0.445 0.337 0.376 0.377 0.376
Average chiasmata
Bivalents
"t" value
Univalents
Rings
Rods
3.21 2.16 2.59 1.98 2.22 2.37 2.93
3.79 4.39 3.88 4.32 4.32 4.09 3.61
1.01 0.74 1.41 0.76 0.85 0.77
10.22 + 9.24 + 10.29 + 8.89 + 9.73 + 9.65 + 10.10 +
1.92 1.92 2.91 3.09 1.61 204 2.86
3.22* 0.105 2.676* 1.944 1.737 0.214
38
Theor. Appl. Genet. 61 (1982)
Fig. 2 a - i. Meiosis in Primary trisomics, a Trivalent association for nucleolar chromosome at pachytene (note the complete pairing of short arms of the three threads), a a explanatory line drawing of 2 A. b Chromosome-3 represented by a bivalent and a univalent with pairing near the centromere region only. b a Explanatory line drawing of 2 B. e Pachytene showing trivalent association for chromosome 5 or 6. c 1 Explanatory line drawing of 2 C. d Diakinesis in triplo-2 showing the extra chromosome attached to the nucleolus, e Diakinesis in triplo-I showing trivalent type 7 and 6ii. f Metaphase I in triplo-3, showing trivalent type 8 and 6ii. g Metaphase I in triplo-1, showing trivalent type-7 in alternate orientation and 6II. h Late anaphase I in trisomic-3 showing unilateral movement by the two chromatids resulting from univalent division, i Telo-I in "Narrow" trisomic, showing the extra chromosome, forming a micronucleus
values were fairly constant. Trivatent formation varied from 8.69% to 54.09% of the cells examined in different classes. There appears to be no relationship between the length of the extra chromosome and the frequency o f trivalent formation as revealed by the chromosome associations in different classes. For example, triplo-3 had a trisomic condition for chromosome-3, which has a total length of 38.04 ~tm (Fig. 2 b). It formed trivalent in 33.32% o f the cells examined, while triplo-1, carrying the longest chromosome-1 (43.40 ~tm) appeared in only 28.44% of the cells as a trivalent. On the other hand, the nucleolar trisomic, with a length o f 39.14~tm, formed a trivalent in a m a x i m u m n u m b e r of cells. In "Slender" trisomic, as many as 25.32% of the cells showed the presence o f one o f the bivalents, represented by a pair of univalents. A similar situation was encountered in other classes, but at lower frequencies. Trivalent types 7, 8 (Fig. 2 e, f) and 9 were observed in all classes studied. The former two were more frequent
than type-9, which was observed rarely. Trisomics for subterminal chromosomes (No. 2, 3) formed particularly low frequency o f trivalent type-9. In view of the frequent formation o f a chain of three in the trisomic for nucleolar chromosome, it appears that the short arm is also involved in chiasma formation, although it was only 5.46 ~tm length. Mean chiasma frequencies recorded in different trisomics indicates that the trisomic for nucleolar chromosome with the highest frequency o f trivalent association also formed an increased n u m b e r o f chiasmata per cell, compared to diploid and other trisomic classes (Table 2). However, no precise relationship between the length of the extra chromosome and the chiasma frequency was observed, since triplo-1, having the longest extra chromosome, formed a lower n u m b e r o f chiasmata than the four other trisomic classes, and triplo-3 having an extra chromosome of 38.04 ~tm, formed the lowest number of chiasmata.
V. R. Reddi and V. Padmaja: Studies on Aneuploids of Petunia. Part 1
39
Anaphase I distribution of 8 to 7 chromosomes was observed in a majority of the cases as expected, and it was at a m a x i m u m (84.41%) in "semi" trisomic. The rest of the time, it was 7 to 7 with a univalent lagging (Fig. 2i). The division of the univalent was observed occasionally, in all the classes of the present study. It was more frequent in trisomic-I (26.04%). The two chromatids, resulting from delayed division, showed late separation. Rarely, as observed in the "slender" trisomic, did the chromatids show unilateral m o v e m e n t (Fig: 2 k). At anaphyse II, most frequent type of chromosomal grouping observed was, 7 + 7 + 8 + 8 followed by 7 + 7 + 7 + 7 with two laggards, which formed themselves into one or two micronuclei. In 1.36% of the cells in "oval" trisomic, 3 of the 4 telophase II nuclei were with 8 chromosomes each, indicating the division of univalent twice.
ma formation, even in a considerable n u m b e r of associations of three at pachytene. It is also likely that cases where non-homologous or non-specific pairing was observed, m a y not lead to chiasma formation at all. No clear relationship between the trivalent frequency and length of extra chromosome could be established in the present study, as has already been pointed out. Similar results with respect to relative chromosome length and trivalent frequency were reported earlier in barley (Tsuchiya 1960), Nicotiana sylvestris (Goodspeed and Avery 1939), Trigonella (Singh and Singh 1976, 1980) and in pearl millet (Manga 1976). On the other hand, longer chromosomes were found to form trivalents at higher frequencies in Jute (Thakare et al. 1974), maize (Einset 1943), tomato (Rick and Barton 1954), Lotus pedunculatus (Chen and Grant 1968a) and in pearl millet (Gill and Virmani 1971). Divergent reports in different varieties of the same species, as in pearl millet, reveal that genotypic differences control chiasma formation in a complicated way.
Discussion
Literature
In the present study, the 7 primary trisomics of Petunia can be recognised from each other with reasonable clarity. This m a y suggest the basic diploid nature o f the genome, taking exception of a few duplications that m a y be present in otherwise non-homologous chromosomes. Bridges (1922) advanced the theory of gene balance and indicated that the imbalance, caused by the addition of a single chromosome, is reflected in physiological disturbance and morphological as well as developmental deviation. In addition, McDaniel and Ramage (1970) have shown specific protein differences in barley, caused by the presence of a specific extra chromosome. Identification of the extra chromosome at pachytene, in 3 of the 7 trisomics in the present study revealed that the three homologous m a y pair to form a trivalent (Fig. 2 a - c ) , or m a y be represented by a bivalent and univalent, generally in the form of a foldback, with interarm pairing. In an association of three, however, two of the three chromosomes pair at a given point along the length of the chromosome. The ambiguity in identification is amplified by the occurrence of non-homologous pairing. Similar non-homologous pairing was also reported in the primary trisomics of tomato (see Khush 1973), Sorghum (Poon and Wu 1967; Venkateswarlu and Reddi 1968) and Solanum (Vogt and Rowe 1968; Ramanna and Wagenvoort 1976). In a few preparations of the nucleolar trisomic, all the three homologues paired in the short arm (Fig. 2 a) as was also reported in the nucleolar trisomics of tomato (Lam and Erickson 1971). In 6 of the 7 trisomics of the present study, the average trivalent frequency ranged from 0.30 to 0.44 per cell. Such a low frequency reflects the failure of chias-
Bridges, C. B. (1922): The origin and variations in sexual and sex limited characters. Am. Nat. 56, 51 - 63 Chen, C.C.; Grant, W.F. (1968a): Morphological and cytological identification of the primary trisomics of Lotus pedunculatus. Canad. J. Genet. Cytol. 10, 161-179 Einset, J. (1943): Chromosome length in relation to transmission frequency of maize trisomics. Genetica 28 (5), 349364 Gill, B.S.; Virmani, S.S. (1971): Triple trisomics in pearl millet. Curr. Sci. 40, 22 Goodspeed, T.H.; Avery, P. (1939): Trisomic and other types in Nicotiana sylvestris. J. Genet. 38 (3), 381- 458 Khush, G.S. (1973): Cytogenetics of Aneuploids. New York, London: Acad. Press Levan, A. (1937): Chromosome numbers in Petunia. Hereditas 23, 99 - 112 Lam, S.L.; Erickson, H.T. (1971): The nucleolar trisomic and trisomic transmission in a diploid potato. J. Hered. 62(b), 375-376 Maizonnier, D. (1976): Production of natural tetraploids and trisomics in P. hybrida. Ann. Amelior Plant. (Paris) 26 (2), 305-318 Manga, V. (1976): Chiasma frequencies in primary trisomics of pearl millet. Canad. J. Genet. Cytol. 15, 11-15 McDaniel, R.G.; Ramage, R.T. (1970): Genetics of a primary trisomic series in barley. Identification by protein electrophoresis. Canad. J. Genet. Cytol. 12, 490-495 Padmaja, D.; Reddi, V.R. (1981): Morphology of pachytene chromosomes in Petunia. J. Cytol. Genet. (in press) Poon, N.H.; Wu, H.K. (1967): Identification of involved chromosomes in trisomics of Sorghum vulgate. Pers. J. Agr. Ass. (China) 58 (N.S), 18-32 Ramanna, M.S.; Wagenvoort, M. 0976): Identification of the trisomics series in diploid Solanum tuberosum L. Group tuberosum. Chromosome identification. Euphytica. 25, 233 to 240 Reddi, V.R.; Padmaja, V. (1980): Chromosome behaviour in triploid Petunia. J. Indian Bot. Soc. (abstracts supplement) 59, 8-20
40 Rick, C.M.; Barton, D.W. (1954): Cytological and genetical identification of the primary trisomics of tomato Genetics. 39, 640- 666 Singh, D.; Singh, A. (1976): Primary trisomics in TrigoneUa Frenum graecum. The Nucleus 19, 91-95 Singh, D.; Singh, A. (1980): Primary trisomics of Trigonella eornieulata. Caryologia. 33, 33-39 Smith, F.J.; De Jong, J.H.; Oud, J.L. (1975): The use of primary trisomics for the localisation of genes on the seven different chromosomes of P. hybrida 1. Triplo V. Genetica 45, 361 - 370 Sullivan, T.D. (1947): Somatic chromosomes of pedigreed Petunia hybrida. Bull. Torrey. Bot. Club. 74, 453-475 Thakare, R.G.; Joshua, D.C.; Rao, N.S. (1976): Radiation induced trisomics in Jute. Indian J. Genet. Plant Breeding 34, 337-345 Tsuchiya, T. (1960): Cytogenetic studies of trisomics in barley. Jpn. J. Bot. 17, 177-213
Yheor. Appl. Genet. 61 (1982) Venkateswarlu, J.; Reddi, V.R. (1968): Cytological studies of Sorghum trisomics. J. Here& 59, 179-182 Vogt, G.E.; Rowe, P.R. (1968): Aneuploids from triploid-diploid crosses in the series tuberosa of the genus Solanum. Canad. J. Genet. Cytol. 10, 479-486 Received April 27, 1981 Communicated by B. R. Murty Prof. Dr. V.R. Reddi Dr. V. Padmaja Department of Botany Andhra University Waltair 530003 AP (India)
9 Spfinger-Vedag1982
Studies on Aneuploids of Petunia Part 1: Cytomorphological Identification of Primary Trisomics V.R. Reddi and V. Padmaja Department of Botany, Andhra University, Waltair 530003 (India)
Summary. The seven possible primary trisomics of Petunia (2 n = 14) located in the progenies of triploid, hypertriploid and hypotriploid plants were distinguished from one another and from diploid on the basis of cytological and morphological criteria. They were provisionally named as "Oval", "Semi", "Slender", "Pseudonormal", "Arrow", "Narrow" and "Giant". In three of the trisomics, the extra chromosome was identified for the first time at pachytene stage. Postpachytene studies revealed no precise relationship between the length of extra chromosome and the frequency of multiple association. Key words: P e t u n i a - Primary trisomics - Morphology - Chromosome associations - Chiasma
Materials and Methods Triploids, which formed the main source for producing trisomics, were derived from the crosses of induced autotetraploids of C2 generation with diploids. They were selfed and crossed reciprocally to normal diploids and sowings were made immediately to raise progenies. Several morphological parameters such as plant height, plant type, leaf shape and size, leaf tip, surface and margin, phyllotaxy, petiole and intemode length were taken into consideration, apart from floral and capsule characters, for classifying the trisomics. Flower buds were fixed in 1:4 acetic alcohol and chromosomal counts for the initial detection of trisomy were made from acetocarmine smears showing diakinesis or metaphase ! stages.
Introduction Results Offspring of triploid, hypertriptoid and hypotriploid plants in the white flowered variety of P. axillaris (Lam.) B.S.P., were examined in order to locate the primary trisomics. Cytological screening of 459 plants representing progenies from 3nX 2n; 2 n • 3n and 4 n x 3 n crosses, revealed 179 to be aneuploid. Although the occurrence of trisomics in the genus has been reported earlier (Levan 1937; Sullivan 1947; Smith et al. 1975; Maizonnier 1976) identification of the trisomic chromosome was made only recently by the application of the fluorescence staining technique to enzymatically macerated root tips (Smith et al. 1975). In the present investigation, it was possible to recognize, apparently for the first time, an extra chromosome in three of the primary trisomics at pachytene, based on recognizable landmarks on the individual chromosomes of the diploid complement (Padmaja and Reddi 1981). This paper describes the results of our first attempt and deals with morphological and cytological characteristics of the seven primary trisomics.
Classification into 7 categories on the basis of mature plant characters was made, which corresponded to the basic number. Leaf shape varied in the different classes, facilitating classification to some extent. Even so, 5 of the 7 trisomics were named after leaf shape, and careful examination was made of all the other characters mentioned in order to provide detailed description of individual classes. The trisomics were named provisionally as "Oval" (Triplo-1), "Semi" (Triplo-2), "Slender" (Triplo-3), "Pseudonormal", "Arrow", "Narrow" and "Giant", and were recovered at different frequencies in the triploid progenies. Meiosis in triploid Petunia revealed that the trisomics owe their origin to the irregular distribution of chromosomes at anaphase I (Reddi and Padmaja 1980). Only 3 of the 7 trisomics could be identifiied at pachytene, while the demarcation between 2 others, "Arrow" and "Narrow", was not certain. A brief description of the morphology of the 7 classes is presented below.
0040-5752/82/0061/0035/$ 1.20
36 Trisomic-I-"Oval". The two cotyledonary leaves, as well as those produced later, were "oval" in shape, with obtuse leaf tips (Fig. 1 b). This is in contrast to the lanceolate shape in the diploids with acute leaf tips (Fig. 1 b). Further, they showed comparatively poor growth rate, erect plant type, with variable stem thickness: Leaves were small, length/breadth ratio less than normal and margin entire, thick and somewhat succulent with short petioles. Capsules, was well as the sepals, were short with obtuse tips, like the leaves themselves. Seed set was almost normal.
Theor. Appl. Genet. 61 (1982) diameter (Fig. ld). Leaves were narrowly lanceolate, tip pointed, margin slightly wavy, dark green in colour, with longer petioles, especially on lower leaves. Flowers of normal size, but with longer pedicels, and purple in colour. Sepals narrow, long and pointed like the leaves themselves. Capsules were long, also with pointed tips. "Pseudonormal". Plants of this trisomic class were in no way different from normal diploids (Fig. 1 e), except for the slow growth rate in the seedling stage. The growth rate was better, however, when compared with plants of other trisomic classes.
Trisomic-2-"Semi". Plants with nucleolar chromosome in trisomic condition and were characterised by broad leaves with acute leaf tips (Fig. 1 c) and thus differ from the pointed leaf tips of the diploids and obtuse tips of Trisomic-1. Length/Breadth ratio was less and they resembled tetraploids in this respect, but, differed from the latter in leaf texture. Capsules did not differ from normal diploids, except for the smaller size.
"Arrow" Identified by the "Arrow"-like shape of the leaves which were characteristically arranged. Erect plant type with medium height, developing 2 - 4 lateral branches; leaves - lanceolate, the width of which gradually decreases from base to top resulting in "Arrow" like shape (Fig. 1 f). The dark green coloured leaves showed compact arrangement, pointed upwards.
Trisomic-3-"Slender'" Plants carrying chromosome-3 in trisomic condition, and were characterised by tall, slender, creeping stem bearing lateral branches, mainly at the distal region which remained thin with reduced
"'Narrow" There was close resemblance between this and "slender" trisomic class. However, the differences were that the leaves of"Narrow" were lanceolate and narrow, consistently yellowish green in colour, stem
Fig. 1 a - h . Morphology of diploid and primary trisomic plants, a Diploid; b Triplo-1 "Oval"; c Triplo-2 "Semi"; d Triplo-3 "Slender"; e "Pseudonormal"; f "Arrow"; g "Narrow"; h "Giant"
37
V.R. Reddi and V. Padmaja: Studies on Aneuploids of Petunia. Part 1 Table 1. Chromosome association in 6 primary trisomics at diakinesis and metaphase I Type of association
Trisomic class
III
"Oval . . . .
II
I
Ring Rod 1 -
PMC's
%
PMC's
1 -
1 1 2 1 3 2 2
7 6 5 6 5 5 4 4 4
1 1 3 1 3 1 3
1
1
4
2
1
1.49
-
1 1 -
3 3 2 4
3 3 3 2
3 2 3
2 5 6 -
2.98 7.46 8.95 -
. 29 19 4
-
4 12 4 . 10 2 15 3 3
Semi . . . .
Total
5.97 17.91 5.97 . . 14.92 2.98 22.38 4.47 4.47
67
Slender . . . .
%
12 11
7.22 6.62
34 40 17 -
20.48 24.09 10.24 -
.
-
.
PMC's
%
PMC's
2 7 4 2 15 5 14 8 4
2.66 9.33 5.33 2.66 20.00 6.66 18.66 10.66 5.33
9 13 . 24 2 27 13 .
1
. 17.46 11.44 2.40
166
Pseudonormal . . . . Arrow . . . .
1.33
.
. 8.00 8.00 1.33
6 6 1 75
thin a n d slender, but n o t so thin as to attain c r e e p i n g habit (Fig. 1 g). F l o w e r s w e r e white in colour, with increased corolla d i a m e t e r , sepals also y e l l o w i s h - g r e e n , n a r r o w a n d long. G o o d seed set was o b s e r v e d o n l y in self p o l l i n a t e d flowers.
"Giant" Trisomic. O n l y two plants w e r e f o u n d with interesting p h e n o t y p e s in w h i c h flower size i n c r e a s e d m a r k e d l y (Fig. 1 h). G r o w t h rate was slow, p l a n t type erect, short m a i n stem w i t h no lateral branches, a n d the leaves w e r e n a r r o w l y l a n c e o l a t e a l o n g the entire m a r gin, o f p o i n t e d tips a n d l o n g pedicels, especially in the lowert ones. T h e y w e r e c o m p a c t l y a r r a n g e d with shorter internodes. O n e o f these plants f l o w e r e d early on 58th day a n d the flowers w e r e larger t h a n in the tetraploids. G o o d seed set was o b t a i n e d , after crossing with diploid, but the h y b r i d seed failed to g e r m i n a t e . Both
. 8 5 -
%
8.91 12.87 . . 23.76 1.98 26.73 12.87 . . . . 7.92 4.95 -
101
PMC's 1 2 3 . 26 2 22 14 3 .
%
PMC's
1.19 2.38 3.57 .
Narrow" %
1 3
1.07 3.22
11 1 33 17 -
11.82 1.07 35.48 18.27
3 9 8 7
3.22 9.67 8.60 7.52
. 30.95 2.38 26.19 16.66 3.57
.
6 4 1
-
. 7.14 4.76 1.19
84
93
plants d i e d for u n k n o w n reasons 2 0 - 2 5 days after flowering, so that n e i t h e r meiosis n o r b r e e d i n g b e h a v i o u r could be studied.
Meiosis in Primary Trisomics. C h r o m o s o m e associations w e r e m a i n l y o f Iil I + 6 . (Fig. 2 e) or 7 . + Ii (Fig. 2 d) as expected. But, in a few cells, u n i v a l e n t s w e r e f o u n d r e p l a c i n g o n e or two bivalents ( T a b l e 1). In a d d i tion, the relative f r e q u e n c y o f ring a n d r o d type o f bivalents, w h i c h t e n d to alter c h i a s m a frequencies, was f o u n d to vary in different trisomic classes ( T a b l e 2). In trisomic-I, d e s i g n a t e d as " O v a l " , the extra chrom o s o m e was p r e s e n t as a u n i v a l e n t in 67.14% o f the cells e x a m i n e d in two plants, while in the rest it f o r m e d a trivalent c o n f i g u r a t i o n ( T a b l e 1). T h e f r e q u e n c i e s o f trivalents a n d u n i v a l e n t s v a r i e d a p p r e c i a b l y in different plants o f the s a m e trisomic class, b u t the a v e r a g e
Table 2. Comparison of frequencies of trivalents, bivalents and univalents in 6 primary trisomics at diakinesis Trisomic class
Diploid control "Oval" "Semi" "Slender" "Pseudonormal" "Arrow" "Narrow"
No. of cells analysed
98 56 166 77 101 61 93
* Significant at 1% level
Average number per cell Trivalents
0.303 0.445 0.337 0.376 0.377 0.376
Average chiasmata
Bivalents
"t" value
Univalents
Rings
Rods
3.21 2.16 2.59 1.98 2.22 2.37 2.93
3.79 4.39 3.88 4.32 4.32 4.09 3.61
1.01 0.74 1.41 0.76 0.85 0.77
10.22 + 9.24 + 10.29 + 8.89 + 9.73 + 9.65 + 10.10 +
1.92 1.92 2.91 3.09 1.61 204 2.86
3.22* 0.105 2.676* 1.944 1.737 0.214
38
Theor. Appl. Genet. 61 (1982)
Fig. 2 a - i. Meiosis in Primary trisomics, a Trivalent association for nucleolar chromosome at pachytene (note the complete pairing of short arms of the three threads), a a explanatory line drawing of 2 A. b Chromosome-3 represented by a bivalent and a univalent with pairing near the centromere region only. b a Explanatory line drawing of 2 B. e Pachytene showing trivalent association for chromosome 5 or 6. c 1 Explanatory line drawing of 2 C. d Diakinesis in triplo-2 showing the extra chromosome attached to the nucleolus, e Diakinesis in triplo-I showing trivalent type 7 and 6ii. f Metaphase I in triplo-3, showing trivalent type 8 and 6ii. g Metaphase I in triplo-1, showing trivalent type-7 in alternate orientation and 6II. h Late anaphase I in trisomic-3 showing unilateral movement by the two chromatids resulting from univalent division, i Telo-I in "Narrow" trisomic, showing the extra chromosome, forming a micronucleus
values were fairly constant. Trivatent formation varied from 8.69% to 54.09% of the cells examined in different classes. There appears to be no relationship between the length of the extra chromosome and the frequency o f trivalent formation as revealed by the chromosome associations in different classes. For example, triplo-3 had a trisomic condition for chromosome-3, which has a total length of 38.04 ~tm (Fig. 2 b). It formed trivalent in 33.32% o f the cells examined, while triplo-1, carrying the longest chromosome-1 (43.40 ~tm) appeared in only 28.44% of the cells as a trivalent. On the other hand, the nucleolar trisomic, with a length o f 39.14~tm, formed a trivalent in a m a x i m u m n u m b e r of cells. In "Slender" trisomic, as many as 25.32% of the cells showed the presence o f one o f the bivalents, represented by a pair of univalents. A similar situation was encountered in other classes, but at lower frequencies. Trivalent types 7, 8 (Fig. 2 e, f) and 9 were observed in all classes studied. The former two were more frequent
than type-9, which was observed rarely. Trisomics for subterminal chromosomes (No. 2, 3) formed particularly low frequency o f trivalent type-9. In view of the frequent formation o f a chain of three in the trisomic for nucleolar chromosome, it appears that the short arm is also involved in chiasma formation, although it was only 5.46 ~tm length. Mean chiasma frequencies recorded in different trisomics indicates that the trisomic for nucleolar chromosome with the highest frequency o f trivalent association also formed an increased n u m b e r o f chiasmata per cell, compared to diploid and other trisomic classes (Table 2). However, no precise relationship between the length of the extra chromosome and the chiasma frequency was observed, since triplo-1, having the longest extra chromosome, formed a lower n u m b e r o f chiasmata than the four other trisomic classes, and triplo-3 having an extra chromosome of 38.04 ~tm, formed the lowest number of chiasmata.
V. R. Reddi and V. Padmaja: Studies on Aneuploids of Petunia. Part 1
39
Anaphase I distribution of 8 to 7 chromosomes was observed in a majority of the cases as expected, and it was at a m a x i m u m (84.41%) in "semi" trisomic. The rest of the time, it was 7 to 7 with a univalent lagging (Fig. 2i). The division of the univalent was observed occasionally, in all the classes of the present study. It was more frequent in trisomic-I (26.04%). The two chromatids, resulting from delayed division, showed late separation. Rarely, as observed in the "slender" trisomic, did the chromatids show unilateral m o v e m e n t (Fig: 2 k). At anaphyse II, most frequent type of chromosomal grouping observed was, 7 + 7 + 8 + 8 followed by 7 + 7 + 7 + 7 with two laggards, which formed themselves into one or two micronuclei. In 1.36% of the cells in "oval" trisomic, 3 of the 4 telophase II nuclei were with 8 chromosomes each, indicating the division of univalent twice.
ma formation, even in a considerable n u m b e r of associations of three at pachytene. It is also likely that cases where non-homologous or non-specific pairing was observed, m a y not lead to chiasma formation at all. No clear relationship between the trivalent frequency and length of extra chromosome could be established in the present study, as has already been pointed out. Similar results with respect to relative chromosome length and trivalent frequency were reported earlier in barley (Tsuchiya 1960), Nicotiana sylvestris (Goodspeed and Avery 1939), Trigonella (Singh and Singh 1976, 1980) and in pearl millet (Manga 1976). On the other hand, longer chromosomes were found to form trivalents at higher frequencies in Jute (Thakare et al. 1974), maize (Einset 1943), tomato (Rick and Barton 1954), Lotus pedunculatus (Chen and Grant 1968a) and in pearl millet (Gill and Virmani 1971). Divergent reports in different varieties of the same species, as in pearl millet, reveal that genotypic differences control chiasma formation in a complicated way.
Discussion
Literature
In the present study, the 7 primary trisomics of Petunia can be recognised from each other with reasonable clarity. This m a y suggest the basic diploid nature o f the genome, taking exception of a few duplications that m a y be present in otherwise non-homologous chromosomes. Bridges (1922) advanced the theory of gene balance and indicated that the imbalance, caused by the addition of a single chromosome, is reflected in physiological disturbance and morphological as well as developmental deviation. In addition, McDaniel and Ramage (1970) have shown specific protein differences in barley, caused by the presence of a specific extra chromosome. Identification of the extra chromosome at pachytene, in 3 of the 7 trisomics in the present study revealed that the three homologous m a y pair to form a trivalent (Fig. 2 a - c ) , or m a y be represented by a bivalent and univalent, generally in the form of a foldback, with interarm pairing. In an association of three, however, two of the three chromosomes pair at a given point along the length of the chromosome. The ambiguity in identification is amplified by the occurrence of non-homologous pairing. Similar non-homologous pairing was also reported in the primary trisomics of tomato (see Khush 1973), Sorghum (Poon and Wu 1967; Venkateswarlu and Reddi 1968) and Solanum (Vogt and Rowe 1968; Ramanna and Wagenvoort 1976). In a few preparations of the nucleolar trisomic, all the three homologues paired in the short arm (Fig. 2 a) as was also reported in the nucleolar trisomics of tomato (Lam and Erickson 1971). In 6 of the 7 trisomics of the present study, the average trivalent frequency ranged from 0.30 to 0.44 per cell. Such a low frequency reflects the failure of chias-
Bridges, C. B. (1922): The origin and variations in sexual and sex limited characters. Am. Nat. 56, 51 - 63 Chen, C.C.; Grant, W.F. (1968a): Morphological and cytological identification of the primary trisomics of Lotus pedunculatus. Canad. J. Genet. Cytol. 10, 161-179 Einset, J. (1943): Chromosome length in relation to transmission frequency of maize trisomics. Genetica 28 (5), 349364 Gill, B.S.; Virmani, S.S. (1971): Triple trisomics in pearl millet. Curr. Sci. 40, 22 Goodspeed, T.H.; Avery, P. (1939): Trisomic and other types in Nicotiana sylvestris. J. Genet. 38 (3), 381- 458 Khush, G.S. (1973): Cytogenetics of Aneuploids. New York, London: Acad. Press Levan, A. (1937): Chromosome numbers in Petunia. Hereditas 23, 99 - 112 Lam, S.L.; Erickson, H.T. (1971): The nucleolar trisomic and trisomic transmission in a diploid potato. J. Hered. 62(b), 375-376 Maizonnier, D. (1976): Production of natural tetraploids and trisomics in P. hybrida. Ann. Amelior Plant. (Paris) 26 (2), 305-318 Manga, V. (1976): Chiasma frequencies in primary trisomics of pearl millet. Canad. J. Genet. Cytol. 15, 11-15 McDaniel, R.G.; Ramage, R.T. (1970): Genetics of a primary trisomic series in barley. Identification by protein electrophoresis. Canad. J. Genet. Cytol. 12, 490-495 Padmaja, D.; Reddi, V.R. (1981): Morphology of pachytene chromosomes in Petunia. J. Cytol. Genet. (in press) Poon, N.H.; Wu, H.K. (1967): Identification of involved chromosomes in trisomics of Sorghum vulgate. Pers. J. Agr. Ass. (China) 58 (N.S), 18-32 Ramanna, M.S.; Wagenvoort, M. 0976): Identification of the trisomics series in diploid Solanum tuberosum L. Group tuberosum. Chromosome identification. Euphytica. 25, 233 to 240 Reddi, V.R.; Padmaja, V. (1980): Chromosome behaviour in triploid Petunia. J. Indian Bot. Soc. (abstracts supplement) 59, 8-20
40 Rick, C.M.; Barton, D.W. (1954): Cytological and genetical identification of the primary trisomics of tomato Genetics. 39, 640- 666 Singh, D.; Singh, A. (1976): Primary trisomics in TrigoneUa Frenum graecum. The Nucleus 19, 91-95 Singh, D.; Singh, A. (1980): Primary trisomics of Trigonella eornieulata. Caryologia. 33, 33-39 Smith, F.J.; De Jong, J.H.; Oud, J.L. (1975): The use of primary trisomics for the localisation of genes on the seven different chromosomes of P. hybrida 1. Triplo V. Genetica 45, 361 - 370 Sullivan, T.D. (1947): Somatic chromosomes of pedigreed Petunia hybrida. Bull. Torrey. Bot. Club. 74, 453-475 Thakare, R.G.; Joshua, D.C.; Rao, N.S. (1976): Radiation induced trisomics in Jute. Indian J. Genet. Plant Breeding 34, 337-345 Tsuchiya, T. (1960): Cytogenetic studies of trisomics in barley. Jpn. J. Bot. 17, 177-213
Yheor. Appl. Genet. 61 (1982) Venkateswarlu, J.; Reddi, V.R. (1968): Cytological studies of Sorghum trisomics. J. Here& 59, 179-182 Vogt, G.E.; Rowe, P.R. (1968): Aneuploids from triploid-diploid crosses in the series tuberosa of the genus Solanum. Canad. J. Genet. Cytol. 10, 479-486 Received April 27, 1981 Communicated by B. R. Murty Prof. Dr. V.R. Reddi Dr. V. Padmaja Department of Botany Andhra University Waltair 530003 AP (India)
