Plant Cell Reports
Plant Cell Reports (1987) 6:109-113
© Springer-Verlag 1987
A comparison of somatic chromosomal instability in tissue culture regenerants from Medicago media Pers. P. Nagarajan 1 and P. D. W a l t o n 2 1 Biotechnology Department, Alberta Research Council, P.O. Box 8330, Postal Station F, Edmonton, Alberta T6H 5X2, Canada 2 Plant Sciences Division, University of Alberta, Edmonton, Alberta T6G 2P5, Canada Received August 18, 1986 / Revised version received October 29, 1986 Communicated by F.Constabel
ABSTRACT
Two c u l t i v a r s (Heinrichs, Beaver) and two breeding lines (Brl, Lel) from Medicago media were cultured in a media protocol consisting of a high concentration 2,4-D induction step. Regenerants were produced from a l l four stocks. Representative samples from each regenerant population along with the corresponding control population were c y t o l o g i c a l l y analyzed for chromosomal and pollen abnormalities. While numerical changes in chromosome numbers were found in a l l regenerant populations, there was considerable v a r i a t i o n between the four stock groups. Heteroploidy was observed for both hypo and hyper aneuploid regenerants, but there were no differences in pollen s t a i n a b i l i t y between hypo and hyper aneuploid regenerants and 'euploid' regenerants. Tissue culture regenerants generally produced a lower pollen s t a i n a b i l i t y percent as compared to control populations grown from seeds. Gross and cryptic changes in chromosomes, or hormonal carry over effects or both were considered causes for poor pollen s t a i n a b i l i t y in tissue culture regenerants. Cytological analyses indicate that the c u l t i v a r might play an important role in the cytological s t a b i l i t y or i n s t a b i l i t y of regenerant populations. E x p l o i t a t i o n of this n a t u r a l l y e x i s t i n g s i t u a t i o n to produce 'euploid' regenerants for f i e l d experiments and to obtain gross cytological s t a b i l i t Y in somaclones is discussed. Key words:
a l f a l f a somaclones, genome s t a b i l i t y , d i c e n t r i c bridges, anaphase disturbance, aneuploidy, heteroploidy.
INTRODUCTION
Plant tissue culture has been used extensively to produce genetic variants in many crop improvement schemes (Larkin and Scowcroft, 1981; Meins, 1983; Shepard et al 1980). Phenotypic and genotypic v a r i a t i o n s have been studied in a l f a l f a tissue culture regenerants (Reisch and Bingham, 1980; Johnson et al 1984). These studies indicate that tissue culture per se produces abundant cytological anomalies a f f e c t i n g both structural and numerical chromosome c o n s t i t u t i o n . Many phenotypic abnormalities have been correlated to cytological anomalies (Groose and Bingham, 1984). Changes in chromosome structure and numher may
Offprint requests to." P. Nagarajan
be considered a common feature in plants by tissue culture (D'Amato, 1977; Evans and Reed, 1981; Constantin, 1981; Bayliss, 1980). They are generally a hinderance to the use of somaclonal v a r i a t i o n for crop improvement unless the objective warrants using chromosome changes. Field tests usually call for the production of abundant regenerants at the 'euploid' l e v e l . Such populations are ideal for comparisons with the control populations, because they possess chromosomes at a s i m i l a r ploidy l e v e l , and so avoiding this source of v a r i a t i o n . There may be three major factors a f f e c t i n g the chromosomal c o n s t i t u t i o n of tissue culture regenerants: 1) the tissue culture media composition, hormones and the time in culture, 2) presence of genetic factors in the cultured plant that control chromosomal i n s t a b i l i t y and 3) an i n t e r a c t i o n of genotype and culture protocol. The present i n v e s t i g a t i o n was undertaken to see i f there were differences among c u l t i v a r s for the production of a c y t o l o g i c a l l y - s t a b l e regenerant population using a single media protocol. The experiment was carried out in a media protocol consisting of a high concentration 2,4-D induction step which these c u l t i v a r s had previously been shown to e x h i b i t good embryogenic response (Nagarajan e__tt a_]_l 1986). MATERIALS AND METHODS
Somaclones were generated as follows. Seeds of Heinrichs, Beaver and Synthetics Rr-I and Le-I (Br-1 and Le-1 were derived from selections of M. media cv. Vernal, Beaver and Roamer) were s t e r i T i { e d for 15 minutes in a solution of 0.1% mercuric chloride and 0.1% sodium lauryl s u l f a t e , then washed three times in s t e r i l e d i s t i l l e d water. Explants from the root and hypocotyl were removed from 20 seedlings a f t e r the seeds were germinated in the dark at 29% for 5-6 days on a 0.6% agar medium containing 0.2% sucrose. Callus formation from roots and hypocotyl explants occurred in the dark at 29°C over a 25-day period using Blaydes medium (Blaydes, 1966) with 2 mg/L of 2,4-D and 2 mg/L k i n e t i n . Details of the subsequent tissue culture protocol for induction, embryo formation and p l a n t l e t conversion are set out in Table 1. For cytological analyses, root tips were collected from rooted shoot tips and pretreated for 24 hours in ice cold water, fixed in 1:3 propionic
IlO alcohol for two to three days and stored in 70% ethanol at 4°C. Hydrolysis was carried out using 1N HCI at 60°C for six minutes, and the root tips were stained in 2% acetocarmine at 4°C in specimen tubes for two to f i v e days. Stained root tips were squashed in 2% aceto-orcein and observed under a Zeiss Photo I I I microscope using 40 x and I00 x phase contrast objectives. Chromosomal counts were made from a minimum of two root tips from a genotype, and over 30 cells were observed for each genotype. Pollen s t a i n a h i l i t y was carried out on those plants which flowered, using a minimum of two flowers for each genotype. Anthers were squashed in 2% acetocarmine to determine the s t a i n a b i l i t y percentage. On the whole, 240 regenerants were c y t o l o g i c a l l y analysed for somatic chromosomal and pollen abnormalities. To ascertain the extent of v a r i a t i o n in the control seed-produced stocks, 50 genotypes of each c u l t i v a r were raised in pots, and shoot t i p s were rooted and processed as set out in the above schedule. Somatic chromosome number and pollen s t a i n a b i l i t y was determined as above. Table I .
Tissue Culture Protocol
Purpose
Schedule
Induction
*SH + 11.2 mg/L 2,4-D + 1.08 mg/L *Kn + 30 g/L sucrose + 8 g/L agar pH 5.95 (4 days at 29°C in dark)
Embryo Formation
RLAYDES (1966) + 2 g/L *YE + 100 mg/L *myol + 30 g/L sucrose + 8 g/L agar pH 5.95 (35-45 days in continuous l i g h t )
Plantlet Conversion
RLAYDES (1966) + 10 g/L sucrose + 6 g/L agar pH 5.90 (20 days in continuous l i g h t )
*Schenk and Hilderbrandt (1972) *KN = kinetin *YE = yeast extract *myol = myo-inositol RESULTS Cytological analysis for numerical chromosome number revealed that a l l four c u l t i v a r s produced both hyper and hypo aneuploids and euploids in varying proportions. 'Heinrichs' produced the highest percentage of aneuploids (64%) followed by 'Beaver' (21%). Rr-1 and Le-1 both produced low and an almost identical percent of aneuploids (10% and 9.4%). Control plants of these c u l t i v a r s did not contain aneuploid plants. The anueploid and the euploid regenerants of 'Heinrichs', Br-1 and 'Beaver', gave a low pollen s t a i n a b i l i t y score as compared to t h e i r seed derived controls. A more extensive reduction in pollen s t a i n a h i l i t y was recorded in 'Heinrichs' and Br-I regenerants as compared to 'Beaver' regenerants (Table 2). Generally, the control population of each of these c u l t i v a r s gave good pollen s t a i n a b i l i t y scores (90% or over - Table 2). 'Heinrichs' regenerants produced abundant plants containing heteroploid chromosome number (Table 2) both at hyper and hypo aneuploid levels.
They also produced a high percentage of hyper aneuploids (12% - Table 2) containing cells at octo and heptaploid chromosome number within a root tip (Fig. 2 & 3). Most aneuploids can be distinguished morphologically b~ their narrow and small leaf characteristics with a slow and stunted growth habit. The polyploids (hexa- and octopolyploid) were more 'stemmy' than 'leafy' with long internodes and 'gigas' characteristics for leaf size and thickness of stem. Many aneuploids failed to flower, and their a b i l i t y to regrow after each cutting was markedly poor. The mortality rate was high in the aneuploid population. DISCUSSION The media protocol that was used in this study consisted of a high concentration 2,4-D induction step (Table i ) . This protocol was chosen, because all four cultivars responded well in earlier studies giving good embryoid production and plantlet conversion (Nagarajan, et al 1986). Also 2,4-D causes chromosomal i n s t a b i l i t y in tissue culture (Torrey, 1977) and this protocol would enable the testing of cultivar response to tissue culture. Following tissue culture, the most unstable c u l t i v a r was 'Heinrichs', producing the highest rate of aneuploidy. Most aneuploid 'Heinrichs' regenerants exhibited the heteroploid condition, producing cell to cell variations in chromosome number. Similarly, heteroploidy occurred in low frequencies in Brl, Lel and Beaver regenerants. Frequently, heteroploid plants contained cells with anaphase laggards (Fig. 4 & 5) and dicentric bridges (Fig. 6, 7 & 8). Ogura (1976) also reported anaphase bridges in cytological chimeras of tobacco regenerants. In this study, some of the dicentric bridges persisted even after cytokinesis had occurred (Fig. 6). All of these cellular abnormalities indicate that heteroploidy is caused by chromosomal elimination (laggards) and unequal distribution during mitotic anaphase, resulting in a hypo or hyper aneuploid condition. Usually, the origin of heteroploidy may be traced back to early embryogenic events from callus tissue. Recent studies in Trifolium repens indicate that in v i t r o somatic embroids are formed both by direct m u l t i c e l l u l a r budding and by proliferation of single cells, the former being more frequent (Maheshwaran & Williams, 1985). Assuming that 'Heinrichs' produces somatic embryoids predominantly hy multicellular budding, then because callus tissue is highly heterogeneous both cytologically and genetically, the occurrence of heteroploidy would be frequent, as was observed. Naturally occurring heteroploid dwarfs have also been reported in Douglas Fir (Rehfeldt et al 1983). Since 'Heinrichs' regenerants produced abundant cytological abnormalities, these stocks w i l l not be suitable for the production of plants at the 'euploid' level. 'Beaver', on the other hand, produced 21% cytological abnormalities, but its in v i t r o embryogenic performance was poor (Nagarajan et al 1986); so that tissue culture regeneration and establishment to recover euploids from 'Beaver' would be laborious. On the other hand, Br-1 and Le-1 gave good embryogenic response and plantlet conversion (Nagarajan et al 1986), and this study established their high cytological s t a b i l i t y since both cultivars produced at least 90% euploids. Thus, they would be ideal for the production of a large number of plants with low cytological i n s t a b i l i t y . Studies conducted on other cultivars also indicate that cytological s t a b i l i t y could be obtained from tissue culture regenerants and protoclones (Kao & Michayluk 1980, and Mazentsev
111 Fig. I.
An 'euploid' regenerant with 2n=4x=32 chromosome number.
Fig. 2.
An 'octoploid'
Fig. 3.
A 'heptaploid' cell 7n=56.
Fig. 4.
A 'heteroploid' regenerant showing chromosomal laggards at telophase.
Fig. 5.
A 'heteroploid' regenerant showing a 'laggard' at anaphase.
Fig. 6.
A 'heteroploid' regenerant with a ' d i c e n t r i c bridge', persistant even a f t e r 'cytokinesis' had occurred.
Fig. 7,
A 'heteroploid' regenerant with a ' d i c e n t r i c ' bridge.
Fig. 8.
Anaphase disturbances showing dicentric bridges.
regenerant 8n=64.
112 Table 2 - Comparison of the percentage of cytological abnormalities found in control and regenerant populations.
CULTIVARS Cytological Condition
Heinrichs Control Regenerants
Control
Percent Hypo-Aneupl oi dy
-
52.6
-
Percent Pollen-Stainability
-
36.5±23.7
-
Percent Hyper-Aneuploidy
-
12.3
-
2.4
3.9
0.00
Percent Pollen-Stainability
-
44.3±24.6
-
33.4
ND
ND
Percent Euploidy
i00
35.1
I00
90.0
Percent Pollen-Stainability
93±5.8 43.4±19.5
BR-1 Regenerants
7.3 48.6±10.6
90±7.5 52.8±15.0
Beaver Control Regenerants
Le-1 Control Regenerants
-
17.7
9.4
-
73.9±11.6
ND
I00
78.4
9 1 ± 6 . 3 82.7±8.1
100 89±4.3
90.6 ND
ND = Not Determined *BR-1 = Brooks-1 *Le-1 = Lethbridge-1
1981) and our studies confirmed t h e i r i n v e s t i g a t i o n s . The pollen s t a i n a b i l i t y of a l l regenerants was low. For the euploid regenerants, this may be due to cryptic chrosmosomal changes or the effect of hormones or cytokinins. Synthetic hormones such as 2,4-D are not readily destroyed by most plant tissues (Torrey, 1977), and t h e i r effects can be profound on the developing pollen grains. In conclusion, the results indicate that: 1)
Cytological s t a b i l i t y may be c u l t i v a r and genotype dependent.
2)
Some c u l t i v a r s are highly stable (Br-1 and Le-1), and others are highly unstable (Heinrichs and Beaver) when the protocol used in these tests is applied.
3)
The heteroploidy found in regenerants may well arise during early embryogenesis by a m u l t i c e l l u l a r pathway.
4)
Reduction in f e r t i l i t y in a regenerant population may be due to gross and cryptic chromosomal abnormalities or effects due to 2,4-D or both.
ACKNOWLEDGEMENTS
Thanks are due to Drs. E.T. Ringham, Professor, University of Wisconsin, B.G. Thompson and D.F. Gerson (ARC) for suggestions and improvement of this manuscript. ~Eknowledgements are also due to Miss T. Malcolm for helping in processing the samples. REFERENCES
i.
Rayliss, M.W. 1980. Chromosomal v a r i a t i o n in plant tissues in culture. I n t . Rev. Cytol. Suppl. IIA:I13-144.
2.
Blaydes, D.F. 1 9 6 6 . Interaction of k i n e t i n and various i n h i h i t o r s in the growth of soybean tissue. Physiol. Plant. 19:748-753. 3, Constantin, M.J. 1981. Chromosomei n s t a b i l i t y in cell and tissue cultures of regenerated plants. Environ. Exp. Rot. 21:359-368. 4. ~'Amato, F. 1977. Cytogenetics of d i f f e r e n t i a t i o n in tissue in cell cultures, p. 343-357. In J. Reinert and Y.P.S. Bajaj (ed.) Applied and fundamental aspects of plant c e l l , tissue and organ culture. Springer-Verlag, B e r l i n . 5. Evans, D.A. and S.M. Reed. 1981. Cytogenetic techniques, p. 213-240. In T.A. Thorpe (ed.) plant tissue culture, methods and applications in a g r i c u l t u r e . Academic Press, New York. 6. Groose, R.W. and E.T. Bfingham. 1984. Variation in plants regenerated from tissue culture of t e t r a p l o i d a l f a l f a heterozygous for several t r a i t s . Crop Sci. 24:655-658. 7. Johnson, L.B., D.L. S t u t e v i l l e , S.E. Schlarbaum and D.Z. Skinner. 1984. Variation in phenotype and chromosome number in a l f a l f a protoclones regenerated from nonmutagenized c a l l i . Crop Sci. 24:948-951. 8. Kao, K.N. and M.R. Michayluk. 1980. Plant regeneration from mesophyll protoplasts of a l f a l f a . Z. Pflanzen Physiol. 96:135-141. 9. Larkin, P.J. and W.R. Scowcroft. 1981. Somaclonal v a r i a t i o n - a novel source of v a r i a b i l i t y from cell cultures for plant improvement. Theor. Appl. Genet. 60:197214. i0. Maheshwaran, G. and E.G. Williams. 1985. Origin and development of somatic embryoids formed d i r e c t l y on immature embryos of T r i f o l i u m repens in v i t r o . Ann. Bot. 56:619-630.
113 11. Meins, F. 1983. Heritable v a r i a t i o n in plant c e l l c u l t u r e . Ann. Rev. Plant Physiol. 34:327-346. 12. Mazentsev, A.V. 1981. Mass regeneration of plants from the c e l l s and protoplasts of lucerne (in Russian). Dokl. Vses. Akad. S. Kh. Nauk im. V.I. Lenina 4:22-23. 13. Nagarajan, P., J.S. McKenzie and P.D. Walton. 1986. Embryogenesis and plant regeneration of Medicago spp. in tissue c u l t u r e . Pl. Cell. Rep. 5:77-80. 14. Ogura, H.K. 1976. Cytological chimeras in o r i g i n a l regenerants from tobacco tissue. Japan. J. Genetics 51:161-174. 15. Rehfeldt, G.E., S.P. Wells and J.Y. Woo. 1983. Chromosomal imbalances in Douglasf i r (Pseudotsuqa menziesii). Can J. Genet. Cytol. 25:113-116.
16. Reisch, B. and E.T. Bingham. 1980. The genetic control of bud formation from callus cultures of d i p l o i d a l f a l f a . Plant Science Letters 20:71-77. 17. Schenk, R.U. and A.C. Hildebrandt. 1972. rledium and techniques f o r induction and growth of monocotyledonous and d i c o t y l e donous plant cell cultures. Can J. Bot. 50:199-204. 18. Shepard, J . F . , D. Bidney and E. Shahin. 1980. Potato protoplasts in crop improvement. Science (Washington, D.C.) 208:17-24. 19. Torrey, J.G. 1977. C y t o d i f f e r e n t i a t i o n in cultured c e l l s and tissues. Hort. Sci. 12(2):138-139.
Plant Cell Reports (1987) 6:109-113
© Springer-Verlag 1987
A comparison of somatic chromosomal instability in tissue culture regenerants from Medicago media Pers. P. Nagarajan 1 and P. D. W a l t o n 2 1 Biotechnology Department, Alberta Research Council, P.O. Box 8330, Postal Station F, Edmonton, Alberta T6H 5X2, Canada 2 Plant Sciences Division, University of Alberta, Edmonton, Alberta T6G 2P5, Canada Received August 18, 1986 / Revised version received October 29, 1986 Communicated by F.Constabel
ABSTRACT
Two c u l t i v a r s (Heinrichs, Beaver) and two breeding lines (Brl, Lel) from Medicago media were cultured in a media protocol consisting of a high concentration 2,4-D induction step. Regenerants were produced from a l l four stocks. Representative samples from each regenerant population along with the corresponding control population were c y t o l o g i c a l l y analyzed for chromosomal and pollen abnormalities. While numerical changes in chromosome numbers were found in a l l regenerant populations, there was considerable v a r i a t i o n between the four stock groups. Heteroploidy was observed for both hypo and hyper aneuploid regenerants, but there were no differences in pollen s t a i n a b i l i t y between hypo and hyper aneuploid regenerants and 'euploid' regenerants. Tissue culture regenerants generally produced a lower pollen s t a i n a b i l i t y percent as compared to control populations grown from seeds. Gross and cryptic changes in chromosomes, or hormonal carry over effects or both were considered causes for poor pollen s t a i n a b i l i t y in tissue culture regenerants. Cytological analyses indicate that the c u l t i v a r might play an important role in the cytological s t a b i l i t y or i n s t a b i l i t y of regenerant populations. E x p l o i t a t i o n of this n a t u r a l l y e x i s t i n g s i t u a t i o n to produce 'euploid' regenerants for f i e l d experiments and to obtain gross cytological s t a b i l i t Y in somaclones is discussed. Key words:
a l f a l f a somaclones, genome s t a b i l i t y , d i c e n t r i c bridges, anaphase disturbance, aneuploidy, heteroploidy.
INTRODUCTION
Plant tissue culture has been used extensively to produce genetic variants in many crop improvement schemes (Larkin and Scowcroft, 1981; Meins, 1983; Shepard et al 1980). Phenotypic and genotypic v a r i a t i o n s have been studied in a l f a l f a tissue culture regenerants (Reisch and Bingham, 1980; Johnson et al 1984). These studies indicate that tissue culture per se produces abundant cytological anomalies a f f e c t i n g both structural and numerical chromosome c o n s t i t u t i o n . Many phenotypic abnormalities have been correlated to cytological anomalies (Groose and Bingham, 1984). Changes in chromosome structure and numher may
Offprint requests to." P. Nagarajan
be considered a common feature in plants by tissue culture (D'Amato, 1977; Evans and Reed, 1981; Constantin, 1981; Bayliss, 1980). They are generally a hinderance to the use of somaclonal v a r i a t i o n for crop improvement unless the objective warrants using chromosome changes. Field tests usually call for the production of abundant regenerants at the 'euploid' l e v e l . Such populations are ideal for comparisons with the control populations, because they possess chromosomes at a s i m i l a r ploidy l e v e l , and so avoiding this source of v a r i a t i o n . There may be three major factors a f f e c t i n g the chromosomal c o n s t i t u t i o n of tissue culture regenerants: 1) the tissue culture media composition, hormones and the time in culture, 2) presence of genetic factors in the cultured plant that control chromosomal i n s t a b i l i t y and 3) an i n t e r a c t i o n of genotype and culture protocol. The present i n v e s t i g a t i o n was undertaken to see i f there were differences among c u l t i v a r s for the production of a c y t o l o g i c a l l y - s t a b l e regenerant population using a single media protocol. The experiment was carried out in a media protocol consisting of a high concentration 2,4-D induction step which these c u l t i v a r s had previously been shown to e x h i b i t good embryogenic response (Nagarajan e__tt a_]_l 1986). MATERIALS AND METHODS
Somaclones were generated as follows. Seeds of Heinrichs, Beaver and Synthetics Rr-I and Le-I (Br-1 and Le-1 were derived from selections of M. media cv. Vernal, Beaver and Roamer) were s t e r i T i { e d for 15 minutes in a solution of 0.1% mercuric chloride and 0.1% sodium lauryl s u l f a t e , then washed three times in s t e r i l e d i s t i l l e d water. Explants from the root and hypocotyl were removed from 20 seedlings a f t e r the seeds were germinated in the dark at 29% for 5-6 days on a 0.6% agar medium containing 0.2% sucrose. Callus formation from roots and hypocotyl explants occurred in the dark at 29°C over a 25-day period using Blaydes medium (Blaydes, 1966) with 2 mg/L of 2,4-D and 2 mg/L k i n e t i n . Details of the subsequent tissue culture protocol for induction, embryo formation and p l a n t l e t conversion are set out in Table 1. For cytological analyses, root tips were collected from rooted shoot tips and pretreated for 24 hours in ice cold water, fixed in 1:3 propionic
IlO alcohol for two to three days and stored in 70% ethanol at 4°C. Hydrolysis was carried out using 1N HCI at 60°C for six minutes, and the root tips were stained in 2% acetocarmine at 4°C in specimen tubes for two to f i v e days. Stained root tips were squashed in 2% aceto-orcein and observed under a Zeiss Photo I I I microscope using 40 x and I00 x phase contrast objectives. Chromosomal counts were made from a minimum of two root tips from a genotype, and over 30 cells were observed for each genotype. Pollen s t a i n a h i l i t y was carried out on those plants which flowered, using a minimum of two flowers for each genotype. Anthers were squashed in 2% acetocarmine to determine the s t a i n a b i l i t y percentage. On the whole, 240 regenerants were c y t o l o g i c a l l y analysed for somatic chromosomal and pollen abnormalities. To ascertain the extent of v a r i a t i o n in the control seed-produced stocks, 50 genotypes of each c u l t i v a r were raised in pots, and shoot t i p s were rooted and processed as set out in the above schedule. Somatic chromosome number and pollen s t a i n a b i l i t y was determined as above. Table I .
Tissue Culture Protocol
Purpose
Schedule
Induction
*SH + 11.2 mg/L 2,4-D + 1.08 mg/L *Kn + 30 g/L sucrose + 8 g/L agar pH 5.95 (4 days at 29°C in dark)
Embryo Formation
RLAYDES (1966) + 2 g/L *YE + 100 mg/L *myol + 30 g/L sucrose + 8 g/L agar pH 5.95 (35-45 days in continuous l i g h t )
Plantlet Conversion
RLAYDES (1966) + 10 g/L sucrose + 6 g/L agar pH 5.90 (20 days in continuous l i g h t )
*Schenk and Hilderbrandt (1972) *KN = kinetin *YE = yeast extract *myol = myo-inositol RESULTS Cytological analysis for numerical chromosome number revealed that a l l four c u l t i v a r s produced both hyper and hypo aneuploids and euploids in varying proportions. 'Heinrichs' produced the highest percentage of aneuploids (64%) followed by 'Beaver' (21%). Rr-1 and Le-1 both produced low and an almost identical percent of aneuploids (10% and 9.4%). Control plants of these c u l t i v a r s did not contain aneuploid plants. The anueploid and the euploid regenerants of 'Heinrichs', Br-1 and 'Beaver', gave a low pollen s t a i n a b i l i t y score as compared to t h e i r seed derived controls. A more extensive reduction in pollen s t a i n a h i l i t y was recorded in 'Heinrichs' and Br-I regenerants as compared to 'Beaver' regenerants (Table 2). Generally, the control population of each of these c u l t i v a r s gave good pollen s t a i n a b i l i t y scores (90% or over - Table 2). 'Heinrichs' regenerants produced abundant plants containing heteroploid chromosome number (Table 2) both at hyper and hypo aneuploid levels.
They also produced a high percentage of hyper aneuploids (12% - Table 2) containing cells at octo and heptaploid chromosome number within a root tip (Fig. 2 & 3). Most aneuploids can be distinguished morphologically b~ their narrow and small leaf characteristics with a slow and stunted growth habit. The polyploids (hexa- and octopolyploid) were more 'stemmy' than 'leafy' with long internodes and 'gigas' characteristics for leaf size and thickness of stem. Many aneuploids failed to flower, and their a b i l i t y to regrow after each cutting was markedly poor. The mortality rate was high in the aneuploid population. DISCUSSION The media protocol that was used in this study consisted of a high concentration 2,4-D induction step (Table i ) . This protocol was chosen, because all four cultivars responded well in earlier studies giving good embryoid production and plantlet conversion (Nagarajan, et al 1986). Also 2,4-D causes chromosomal i n s t a b i l i t y in tissue culture (Torrey, 1977) and this protocol would enable the testing of cultivar response to tissue culture. Following tissue culture, the most unstable c u l t i v a r was 'Heinrichs', producing the highest rate of aneuploidy. Most aneuploid 'Heinrichs' regenerants exhibited the heteroploid condition, producing cell to cell variations in chromosome number. Similarly, heteroploidy occurred in low frequencies in Brl, Lel and Beaver regenerants. Frequently, heteroploid plants contained cells with anaphase laggards (Fig. 4 & 5) and dicentric bridges (Fig. 6, 7 & 8). Ogura (1976) also reported anaphase bridges in cytological chimeras of tobacco regenerants. In this study, some of the dicentric bridges persisted even after cytokinesis had occurred (Fig. 6). All of these cellular abnormalities indicate that heteroploidy is caused by chromosomal elimination (laggards) and unequal distribution during mitotic anaphase, resulting in a hypo or hyper aneuploid condition. Usually, the origin of heteroploidy may be traced back to early embryogenic events from callus tissue. Recent studies in Trifolium repens indicate that in v i t r o somatic embroids are formed both by direct m u l t i c e l l u l a r budding and by proliferation of single cells, the former being more frequent (Maheshwaran & Williams, 1985). Assuming that 'Heinrichs' produces somatic embryoids predominantly hy multicellular budding, then because callus tissue is highly heterogeneous both cytologically and genetically, the occurrence of heteroploidy would be frequent, as was observed. Naturally occurring heteroploid dwarfs have also been reported in Douglas Fir (Rehfeldt et al 1983). Since 'Heinrichs' regenerants produced abundant cytological abnormalities, these stocks w i l l not be suitable for the production of plants at the 'euploid' level. 'Beaver', on the other hand, produced 21% cytological abnormalities, but its in v i t r o embryogenic performance was poor (Nagarajan et al 1986); so that tissue culture regeneration and establishment to recover euploids from 'Beaver' would be laborious. On the other hand, Br-1 and Le-1 gave good embryogenic response and plantlet conversion (Nagarajan et al 1986), and this study established their high cytological s t a b i l i t y since both cultivars produced at least 90% euploids. Thus, they would be ideal for the production of a large number of plants with low cytological i n s t a b i l i t y . Studies conducted on other cultivars also indicate that cytological s t a b i l i t y could be obtained from tissue culture regenerants and protoclones (Kao & Michayluk 1980, and Mazentsev
111 Fig. I.
An 'euploid' regenerant with 2n=4x=32 chromosome number.
Fig. 2.
An 'octoploid'
Fig. 3.
A 'heptaploid' cell 7n=56.
Fig. 4.
A 'heteroploid' regenerant showing chromosomal laggards at telophase.
Fig. 5.
A 'heteroploid' regenerant showing a 'laggard' at anaphase.
Fig. 6.
A 'heteroploid' regenerant with a ' d i c e n t r i c bridge', persistant even a f t e r 'cytokinesis' had occurred.
Fig. 7,
A 'heteroploid' regenerant with a ' d i c e n t r i c ' bridge.
Fig. 8.
Anaphase disturbances showing dicentric bridges.
regenerant 8n=64.
112 Table 2 - Comparison of the percentage of cytological abnormalities found in control and regenerant populations.
CULTIVARS Cytological Condition
Heinrichs Control Regenerants
Control
Percent Hypo-Aneupl oi dy
-
52.6
-
Percent Pollen-Stainability
-
36.5±23.7
-
Percent Hyper-Aneuploidy
-
12.3
-
2.4
3.9
0.00
Percent Pollen-Stainability
-
44.3±24.6
-
33.4
ND
ND
Percent Euploidy
i00
35.1
I00
90.0
Percent Pollen-Stainability
93±5.8 43.4±19.5
BR-1 Regenerants
7.3 48.6±10.6
90±7.5 52.8±15.0
Beaver Control Regenerants
Le-1 Control Regenerants
-
17.7
9.4
-
73.9±11.6
ND
I00
78.4
9 1 ± 6 . 3 82.7±8.1
100 89±4.3
90.6 ND
ND = Not Determined *BR-1 = Brooks-1 *Le-1 = Lethbridge-1
1981) and our studies confirmed t h e i r i n v e s t i g a t i o n s . The pollen s t a i n a b i l i t y of a l l regenerants was low. For the euploid regenerants, this may be due to cryptic chrosmosomal changes or the effect of hormones or cytokinins. Synthetic hormones such as 2,4-D are not readily destroyed by most plant tissues (Torrey, 1977), and t h e i r effects can be profound on the developing pollen grains. In conclusion, the results indicate that: 1)
Cytological s t a b i l i t y may be c u l t i v a r and genotype dependent.
2)
Some c u l t i v a r s are highly stable (Br-1 and Le-1), and others are highly unstable (Heinrichs and Beaver) when the protocol used in these tests is applied.
3)
The heteroploidy found in regenerants may well arise during early embryogenesis by a m u l t i c e l l u l a r pathway.
4)
Reduction in f e r t i l i t y in a regenerant population may be due to gross and cryptic chromosomal abnormalities or effects due to 2,4-D or both.
ACKNOWLEDGEMENTS
Thanks are due to Drs. E.T. Ringham, Professor, University of Wisconsin, B.G. Thompson and D.F. Gerson (ARC) for suggestions and improvement of this manuscript. ~Eknowledgements are also due to Miss T. Malcolm for helping in processing the samples. REFERENCES
i.
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2.
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