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Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol. 21 n. 1 São Paulo Mar. 1998

http://dx.doi.org/10.1590/S1415-47571998000100016 

Meiosis of anther culture regenerants in asparagus (Asparagus officinalis L.)

 

Leonardo Galli 1, Judith Viégas 1, Eliane Augustin 2, Marcia Ines Eckert 1 and João Baptista da Silva 3
1 Laboratório de Biologia Celular CPACT/UFPel, Departamento de Zoologia e Genética, Instituto de Biologia, Universidade Federal de Pelotas (UFPel), Caixa Postal 354, 96010-900 Pelotas, RS, Brasil.
Send correspondence to J.V.
2 Centro de Pesquisa Agropecuária de Clima Temperado (CPACT), EMBRAPA, Pelotas, RS, Brasil.
3 Departamento de Matemática, Estatística e Computação, Instituto de Física e Matemática, UFPel, Pelotas, RS, Brasil.

 

 

ABSTRACT

Pollen mother cells obtained from regenerated plants of asparagus (Asparagus officinalis L.), in a population composed exclusively of male plants, through the process of anther culture from the hybrid G27 X 22-8, were analyzed during meiosis. It was observed that, during theprocess of anther culture by organogenesis, the pollen mother cells of the regenerants had great genomic instability, as evidenced by disturbances in all the meiotic phases of the first and second division. Furthermore, structural chromosomal abnormalities, in addition to aneuploidy and polyploidy, were observed.

 

 

INTRODUCTION

In asparagus breeding, besides problems due to dioecism and perennial habit, it is well known that male plants, with genotype Mm, are more productive than female plants, with genotype mm (Sneep, 1953). Thus, breeders try to obtain hybrids composed solely of male plants. The traditional method to obtain such hybrids uses homozygous males, with genotype MM (called "supermale"), in the crosses, but their occurrence in nature is very low, since they result from the selfing of hermaphrodite flowers of andromonoecious plants. These andromonoecious plants, that yield both male and hermaphrodite flowers, comprise only 2% of the asparagus male plant population(Randall and Rick, 1941).

Another way of obtaining "supermale" plants is doubling haploids with M genotype. In nature, such haploids may be obtained by polyembryonic pheno- mena, which produce twin plantlets of asparagus with varied ploidies (93% diploids and 7% of other ploidies, includinghaploids).Besidesthelowfrequency, there is a high mortality rate. Therefore, polyembryonic haploids are a rare phenomenon (0.75 to 1.79%) in asparagus populations (Randall and Rick, 1941 and Thévenin, 1968).

Anther culture is a better way to obtain haploids that can be doubled either through colchicine treatment or spontaneously, as an artifact of the method itself, allowing the selection of homozygous males. Fernandes (1987) reported that another advantage of haploids is hemizygous genomic expression, which permits detection of spontaneous mutations. However, the routine use of anther culture in asparagus still presents some problems.

The possibility of obtaining asparagus regenerants from anther diploid tissue is reported by Doré (1975), though such genotypes usually will not be useful for breeding purposes as no MM plants are obtained. In apples, Zhang and Lespinasse (1992) reported that there are two kinds of callus formation: the first originates from anther somatic tissue, which, sometimes, will inhibit the second kind of callus formation, from the haploid microspore. Doré (1975) stated that the presence of female plants in a population of asparagus regenerated from anther culture pre- sumed the microspore origin of these regenerants.

Another problem is the occurrence of soma-clonal or gametoclonal variation, which may result in polyploidy. Obtaining haploid plants is not always an easy task, due to genomic doubling in the first stages of the process (Dunwell, 1985).

 

MATERIAL AND METHODS

Regenerated plants of asparagus (Asparagus officinalis L.) from a population composed exclusively of male plants, obtained by anther culture from the hybrid G27 X 22-8, were transplanted in rows at the Centro de Pesquisa Agropecuária de Clima Temperado (CPACT), Empresa Brasileira de Pesquisa Agro- pecuária (EMBRAPA), Pelotas, RS, Brazil, in 1989. Each row comprised plants originated from a single callus (Bobrowski, 1992). Vigor differences were responsible for the variation of one to nine plants in each row.

A total of 91 regenerant plants from 48 rows were analyzed. Normal meiosis was studied in two plants obtained by micropropagation from the hybrid G27 X 14, from the CPACT’s breeding program.

Flower buds were collected from 9:30 to 10:30 a.m. and immediately fixed in acetic alcohol (3:1) for pollen mother cells (PMC) cytogenetic analysis. The fixative was continuously changed until the green buds became discolored, after which they were kept in a refrigerator, immersed in 70% alcohol.

Before microscopic examination, the buds were hydrolyzed in 5 N HCl for 3 min, at room temperature; the anthers were removed from the buds and indi- vidually squashed with a mixture of propionic carmine (1%) and chloral hydrate (1 ml/1 drop), covered with a cover glass and heated.

At diakinesis, the number of homologous groups (HG) was counted, if possible, in 20 PMC randomly selected from each plant. Cells of the other meiotic phases were observed as they emerged on the slide field, and abnormalities were analyzed.

The differences in the number of HG in 82 regenerants, compared to normal control plants, were statistically analyzed (ANOVA) and compared by the Duncan and Dunnett tests.

 

RESULTS AND DISCUSSION

At diakinesis the cells of control plants showed 10 bivalents according to the diploid basic number of 2n = 2x = 20 for asparagus (Figure 1A), whereas in the 82 regenerants the bivalents were not clearly identifiable, enabling only the visualization of discrete groups, called HG. Therefore, the number of chromosomes involved in each HG was not determined. The regenerants showed PMC with an interplant variation ranging from 7 to 37 HG (Figure 1B). An intercell variation often occurred in the same plant; for example, two regenerants had cells with a difference of 22 HG, plant P20 with cells varying from 10 to 32 HG and plant P58 varying from 14 to 36, whereas plant P46 showed the lowest difference between cells, only 4 HG. In this case the plant had cells with 8 to 12 HG.

 

Ms1650f1.gif (19182 bytes)

Figure 1 - Asparagus officinalis pollen mother cells in diakinesis: A, control plant, and B, anther culture regenerant.

 

The averages of HG from the regenerants (Table I) varied significantly (ANOVA) (P < 0.01). Only two plants (P39 and P46) did not differ from the control (Dunnett test a = 0.01), which suggests that they are diploids.

The results support the hypothesis that there is meiotic instability, based on chromosome pairing which results in univalents, bivalents, trivalents, tetravalents, etc. The occurrence of PMC with more than 20 HG in 59 regenerant plants represents clear evidence of an increase in the chromosome number.

Our results agree with those cited by Sybenga (1992) and obtained by Loidl (1990), who mentioned the occurrence of several levels of chromosome pairing in polyploid plants. Our results also agree with Lazarte and Palser (1976) and Ammal and Kaul (1967), who mentioned instability in the pairing of homologous chromosomes in diploid, triploid and tetraploid asparagus.

We observed chromosomes which will probably be lost or form micronuclei (Figure 2C) in eight of 29 regenerants analyzed in metaphase I. Similar behavior was noticed by Malnassy and Ellison (1970), who analyzed tetraploid regenerants of asparagus derived from root culture via callus formation.

 

Ms1650f2.gif (26218 bytes)

Figure 2 - Asparagus officinalis pollen mother cells in metaphase I: A, control plant; B, anther culture regenerant with larger metaphasic plate;C, anther culture regenerant chromosome out of the equatorial plate.

 

Although it was not possible to visualize individual chromosomes, regenerants often had metaphase plates that were larger than those of normal plants, suggesting that they had more genetic material (Figure 2A and B). The enlargement of regenerant genetic material was followed by variability in size of some PMC, even within the same plant.

 

Table I - Mean number of homologous groups (HG) of the chromosomes in diakinesis from anther culture regenerated plants of asparagus (Asparagus officinalis L.). Number of repetitions = 20, except where indicated.

Plant number Mean HG Duncan test
P4 26.5 A
P25 25.1 AB
P10 24.8 ABC
P68 24.4 ABC
P74* 24.2 ABC
P1 24.2 ABC
P50 23.8

BCD

P26 22.7

BCD

P27 22.7

BCD

P42 22.5

CDE

P6 21.6

DEF

P14 21.4

DEF

P41 20.7

DEFG

P60* 20.4

DEFGH

P3 20.2

EFGH

P29 20.1

EFGH

P57 20.0

EFGH

P22 19.9

FGH

P19 19.9

FGH

P70 19.9

FGH

P52 19.9

FGH

P8 19.7

FGH

P48 19.7

FGH

P23 19.4

FGH

P20 18.9

GHI

P72 18.4

GHIJ

P40 18.3

GHIJK

P80 18.3

GHIJK

P13 18.3

GHIJK

P58 18.1

GHIJK

P55 17.9

GHIJKL

P12 17.8

GHIJKLM

P32 17.6

GHIJKLMN

P51 17.6

GHIJKLMNO

P11 17.5

HIJKLMNO

P62 17.2

HIJKLMNO

P5 17.0

HIJKLMNO

P9 16.9

IJKLMNO

P71 16.9

IJKLMNO

P61 16.7

IJKLMNOP

P64 16.4

JKLMNOPQ

P49 16.2

JKLMNOPQR

P53 16.2

JKLMNOPQR

P18 16.1

JKLMNOPQRS

P30 16.0

JKLMNOPQRS

P24 15.9

JKLMNOPQRS

P76 15.8

KLMNOPQRS

P2 15.8

KLMNOPQRS

P17 15.8

KLMNOPQRS

P7 15.6

LMNOPQRST

P65 15.5

LMNOPQRSTU

P75 15.4

MNOPQRSTUV

P66 15.3

NOPQRSTUVW

P79 15.2

NOPQRSTUVW

P15 15.2

NOPQRSTUVW

P21 15.0

OPQRSTUVW

P28 14.9

OPQRSTUVW

P69 14.9

OPQRSTUVW

P56 14.7

OPQRSTUVWX

P43 14.4

PQRSTUVWXY

P81 14.3

PQRSTUVWXY

P73 14.2

QRSTUVWXYZ

P16 14.2

QRSTUVWXYZ

P44 14.0

RSTUVWXYZ[

P54 13.9

RSTUVWXYZ[

P78 13.9

RSTUVWXYZ[\

P67 13.7

STUVWXYZ[\

P45 13.4

TUVWXYZ[\

P59 13.3

UVWXYZ[\

P33 13.2

VWXYZ[\

P82 13.1

WXYZ[\

P34 12.6

XYZ[\

P31 12.4

YZ[\

P37 12.4

YZ[\

P47 12.4

YZ[\

P38 12.2

Z[\

P77 12.2

Z[\

P63 12.2

Z[\

P36 12.1

[\

P35 11.8

\

P39 10.1+

]

P46 10.1+

]

Control 10.0

]

Means followed by the same letter (or symbol) within the columns do not differ from one another, according to Duncan’s test (a = 0.01).
+not significantly different from control (unilateral Dunnett test, a = 0.01). All other values were significantly different.
*Number of repetitions = 19 for P74 and 8 for P60.

 

Anaphase I showed laggards in 28 plants and bridges in four plants among 37 regenerants analyzed. Often, structural aberrations were clearly suggested by the presence of bridges, and probably aneuploidy, caused by laggard chromosomes. These were present in 30 to 100% of cells of regenerants, at a frequency of one to six laggards per cell. These laggard chromosomes were either lost or located at the edge of the cytoplasm (Figure 3B and C). Ammal and Kaul (1967) mentioned the loss of induced tetraploidy in asparagus caused by similar occurrences.

 

Ms1650f3.gif (24399 bytes)

Figure 3 - Asparagus officinalis pollen mother cells in anaphase I: A, control plant; B and C, anther culture regenerants with laggards.

 

A total of 37 regenerants were analyzed at telophase I. The presence of laggards was evidenced in 16 plants, ranging from 4 to 67%, at a frequency of one to eight laggards per cell. Chromosomal bridges were observed less frequently.

Meiosis II was hardly found, probably due to the fact that this step is shorter than meiosis I (Flory Jr., 1932). Metaphase II of 14 regenerants showed most of the cells with parallel spindles, characteristic of normal meiosis in asparagus, but abnormalities were some- times found (Figure 4).

 

Ms1650f4.gif (23469 bytes)

Figure 4 - Asparagus officinalis pollen mother cells in metaphase II: A, control plant; B, anther culture regenerant with larger metaphasic plate; C, abnormal spindles.

 

In anaphase II, laggards were found in three plants, where the observation of this phase was possible (Figure 5). Bridges, in two of them, were also observed. In telophase II, laggards were evident in eight of the 10 regenerants analyzed.

 

Ms1650f5.gif (24477 bytes)

Figure 5 - Asparagus officinalis pollen mother cells in anaphase II: A, control plant; B, anther culture regenerant; C,anther culture regenerant with laggards.

 

Increased chromosome content was evident in all of the meiotic phases and, in most cases, was followed by enlargement of cell volume. Bajaj (1990), in his review of somaclonal variation, found polyploidy, aneuploidy and mixoploidy in a large number of species.Eckert et al. (1993) detected cells with more than 80 chromosomes in calli of asparagus derived from anther culture, while the normal chromosome number is 2n = 20. These findings conform with our results, that showed regenerant cells with 37 homologous groups that, even if they were all univalents, would amount to 17 extra chromosomes.

Finally, the results permit us to conclude that, during theprocess of regeneration from anther culture callus, the PMC of our particular regenerants showed high genomic instability, evidenced by instabilities in the meiotic phases. Furthermore, the process originated structural chromosomal abnormalities in addition to aneuploidy and polyploidy.

 

ACKNOWLEDGMENTS

The authors are indebted to Prof. Maria da Graça Martino Roth, Andreia Neugebauer Barraz, Vilma Ruas da Silva, and Beatriz Viégas Faria. We are also thankful for the support given by DZG-IB/UFPel, CPACT/EMBRAPA, CAPES, CNPq and FAPERGS.

 

 

RESUMO

Foi analisada a meiose em células mãe de pólen de plantas de aspargo (Asparagus officinalis L.) de uma população composta exclusivamente de plantas masculinas, obtidas através do processo de cultura de anteras do híbrido G27 X 22-8. Foi observado que, durante o processo de cultura de anteras, via calogênese, as células mãe de pólen dos regenerantes apresentaram grande instabilidade genômica, evidenciada por irregularidades nas fases de diacinese, assim como de metáfase, anáfase, telófase da primeira e segunda divisão meiótica. Além disto, o processo originou anormalidades cromossômicas estruturais em adição às aneuploidias e poliploidias.

 

 

REFERENCES

Ammal, E.J. and Kaul, B.L. (1967). Cytomorphological studies in autotetraploidy Asparagus officinalis L. In: Annoted Bibliography on Asparagus (Asparagus officinalis L.) (Hung, L., ed.). National Taiwan University, Taiwan.         [ Links ]

Bajaj, Y.P.S. (1990). Somaclonal Variation in Crop Improvement. I. Springer-Verlag, Berlin.         [ Links ]

Bobrowski, V.L. (1992). Obtenção de plantas de aspargo (Asparagus officinalis L.) através da cultura de anteras. Master’s thesis, Universidade Federal de Pelotas, Pelotas, RS, Brasil.         [ Links ]

Doré, C. (1975). Utilization de la culture in vitro chez l’asperge cultivée. Ph. D. thesis, Faculté Science d’Orsay, Orsay, France         [ Links ]

Dunwell, J.M. (1985). Haploid cell culture. In: Plant Cell Culture, a Practical Approach (Dixon, R.A., ed.) IRL Press, Oxford.         [ Links ]

Eckert, M.I., Scur, L. and Viégas, J. (1993). Variação cromossômica em calos de aspargo (Asparagus officinalis L.), dados preliminares. Rev. Bras. Genet. 16: 188 (Abstract).         [ Links ]

Fernandes, M.I.B. de M. (1987). Perspectivas da biotecnologia para o melhoramento de plantas. Pesq. Agropec. Bras. 22: 881-896.         [ Links ]

Flory Jr., W.S. (1932). Genetics and cytological investigations on Asparagus officinalis L. Genetics 17: 432-437.         [ Links ]

Lazarte, J.E. and Palser, B.F. (1976). Morphology vascular anatomy and embryology of pistillate and staminate flowers of Asparagus officinalis. Am. J. Bot. 66: 753-764.         [ Links ]

Loidl, J. (1990). The initiation of mitotic chromosome pairing: the cytological view. Genome 33: 759-778.         [ Links ]

Malnassy, P. and Ellison, J.H. (1970). Asparagus tetraploidsfrom callus tissue. HortScience 5: 444-445.         [ Links ]

Randall, T.E. and Rick, C.M. (1941). Preliminary cytogenetic studies on polyembriony in Asparagus officinalis L. In: Annoted Bibliography on Asparagus (Asparagus officinalis L.) (Hung, L. ed.). National Taiwan University, Taiwan.         [ Links ]

Sneep, J. (1953). The significance of andromonoecy for the breeding of Asparagus officinalis L. Euphytica 2: 89-95.         [ Links ]

Sybenga, J. (1992). Cytogenetics in Plant Breeding. Springer-Verlag, New York.         [ Links ]

Thévenin, L. (1968) Les problémes d’amélioration chez Asparagus officinalis L. Ann. Amélior. Plant. 18: 327-365.         [ Links ]

Zhang, Y.X. and Lespinasse, Y. (1992). Haploid. In:Biotechnology of Perennial Fruit Crops (Hammerschlag, F.A. and Litz, R.E., eds.). C-A-BInternational, England.         [ Links ]

 

 (Received August 31, 1995)

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