BREEDING, CULTIVARS, ROOTSTOCKS, & GERMPLASM RESOURCES HORTSCIENCE 37(6):959–961. 2002.
An Improved Method for Examining Meiotic Chromosomes in Musa L. Martina T.V. Adeleke, Michael Pillay1, and Bosa E. Okoli2 Crop Improvement Division. Plantain and Banana Improvement Project, International Institute of Tropical Agriculture, Eastern and Southern Africa Regional Center, P.O. Box 7878, Kampala, Uganda Additional index words. banana, plantain, meiosis, cytogenetics Abstract. Meiotic studies in Musa L. have been hampered by: 1) time-consuming efforts required to find the correct stages of cell division; 2) rigidity of the microsporocyte cell wall that makes preparation of smears difficult; and 3) poor staining of prophase chromosomes. This study describes an improved technique to examine meiosis in Musa. The procedure involves dissection of microsporocytes from the anthers, centrifugation to obtain large number of microsporocytes, enzymatic digestion of cell walls and treatment of cells with acetic-alcohol that results in spontaneous bursting of the protoplasts and release of chromosomes. Previous meiotic studies in Musa used acetocarmine that stained only highly condensed metaphase and anaphase chromosomes easily but not the relaxed prophase stages. In this study, we found that silver nitrate, Giemsa and Leishmans’ stain were also effective for staining Musa chromosomes. Silver staining was most effective for the less contracted prophase chromosomes. By providing an improved procedure to examine all the meiotic stages in Musa, this technique will be useful to develop pachytene karyotypes, characterize new hybrids and identify nuclear restitution mechanisms that are important in breeding schemes. Banana and plantain (Musa sp.) are important fruit crops in the tropical and subtropical regions of the world (Robinson, 1996). Most banana cultivars and all plantains have a triploid chromosome number of 2n = 3x = 33 (Cheesman and Larter, 1935), although diploid (2n = 2x = 22) and tetraploid (2n = 4x = 44) clones also exist. A range of cellular and molecular biology tools is being used for the improvement of banana and plantain (Crouch et al., 1998; Pillay et al., 2002). However, there is need for more information on the basic biology of the plant, especially in relation to its cytology. Although a preliminary karyotype has been reported (Dantas et al., 1993), detailed karyotypes have not been established for any of the species in the genus Musa. Early cytological studies in Musa were focused on determination of chromosome numbers (Cheesman, 1932a, 1932b; Cheesman and Larter, 1935). Thereafter, assessing chromosome pairing behavior in some of the first artificially produced triploid × diploid hybrids became the prime activity (Cheesman, 1932a, 1932b; Cheesman and Dodds, 1942). Shepherd (1999) carried out the most comprehensive work on the meiotic behavior of Musa chromosomes. However, this work focused on Received for publication 23 Apr. 2001. Accepted for publication 15 Jan. 2002. We thank A. Tenkouano for maintaining the germplasm bank from which the material for this study was obtained. Support for this research was provided, in part, by the Directorate General for International Cooperation (DGIC ex BADC) Belgium. 1 To whom reprint requests should be addressed. International Mailing address: IITA, c/o. L.W. Lambourn & Co., Carolyn House, 26 Dingwall Road, Croydon CR9 3EE, England E-mail address:
[email protected] 2 Dept. of Plant Science and Biotechnology, Univ. of Port Harcourt, Port Harcourt, Nigeria.
metaphase and anaphase stages of meiosis and provides no information on prophase stages, perhaps, due to difficulties in staining the chromosomes. The individual chromosomes in banana and plantain have not been identified and numbered. This is ascribed mainly to problems associated with preparation of good chromosome spreads, and the small size and poor staining ability of Musa chromosomes (Osuji et al., 1996). Although novel methods to prepare slides for mitotic chromosomes in Musa have been established (Dolezel et al., 1998; Pillay and Adeleke, 2001), only the satellited chromosomes are easily distinguishable. In situ hybridization studies were able to distinguish the genomic constitution of Musa lines (Osuji et al., 1997) and physically map the 18S-25S and 5S ribosomal RNA genes (Dolezel et al., 1998; Osuji et al., 1998) in mitotic chromosomes. Meiotic studies in Musa are complicated by: 1) time-consuming efforts of finding the correct stages of cell division; 2) the rigid cell wall of the microsporocytes that makes preparation of smears difficult; and 3) poor staining ability of prophase chromosomes. This paper describes a protocol for obtaining microspore mother cells representing all stages of meiosis. The procedure is based on protoplast isolation from microspore mother cells after enzyme digestion of the cell wall. We also demonstrate for the first time the effectiveness of silver staining for prophase chromosomes in Musa. Materials and Methods Plant materials. The genetic material used in this study included M. acuminata Colla, ‘Calcutta 4’ (AA), M. balbisiana Colla (BB)
‘Bobby Tannap’ (AAB), ‘Bluggoe’ (ABB), and ‘Ngern’ (AAAB). Genetic materials were selected to represent different genomic combinations within the genus Musa and included both wild and cultivated genotypes. The genotypes are maintained in a germplasm bank at the IITA High Rainfall Station at Onne, in southeastern Nigeria. Selection of anthers. Young male buds were harvested on sunny days between 11:00 AM and 12:00 PM. Bracts were carefully removed to expose the clusters of male flowers. Anthers were dissected from the floral buds and the contents examined for stages of cell division by acetocarmine staining. Anthers with an easily extricable fluid were discarded because they contained either mature pollen grains or tetrads, while those in which the contents were easily extruded were either premeiotic or meiotic stages. Microspore mother cells with meiotic stages generally appeared in floral buds that ranged from 9 to 15 mm in length, although this length is genotype-dependent. Fixation of cells and enzyme digestion. Selected buds were fixed in 3 ethanol : 1 acetic acid solution with 1% ferric chloride as a mordant for 18 to 24 h at 4 °C. Five to six anthers were placed side-by-side on a glass slide and both ends of the anthers were cut off with a sharp razor blade. To prevent the anthers from drying, they were covered in a few drops of LB01 buffer (15 mM Tris, 2 mM NaEDTA, 80 mM KCl, 0.5 mM spermine, 15 µM mercaptoethanol, 0.1% Triton X-100, pH 7.5; Dolezel et al., 1998). The microsporocytes were squeezed out of both ends of the anther with a dissecting needle. The clumps of microsporocytes in suspension were transferred to a microcentrifuge tube and centrifuged at 1000× g for 5 min. The supernatant was discarded and the pellet was resuspended in cold citrate buffer (0.1 M sodium citrate and 0.1 M citric acid, pH 4.5), and the procedure repeated. The pellet was resuspended in 0.2 mL of an enzyme mixture (5% cellulase— Sigma Chemicals, 1% pectinase and 1% pectolyase Y23—Karlan Research, Santa Rosa Calif.) prepared in 0.01 M sodium citrate buffer consisting of 0.01 M sodium citrate and 0.01 M citric acid pH 4.5, and incubated at 37 °C for 30 to 60 min. The cells were pelleted from the enzyme solution, washed by centrifugation in citrate buffer and finally resuspended in ice-cold 70% ethanol. Slide preparation. A drop of the protoplast suspension was placed on a clean slide with a Pasteur pipette and left to air-dry. Shortly before the smear dried completely, a drop or two of freshly prepared 3 ethanol : 1 acetic acid was placed over the cells. The quality of the slide was assessed by phase contrast microscopy. Selected slides were air dried and stained with: 1) freshly mixed Giemsa stain (Sigma, U.S.A.) – 3 mL stock solution Giemsa (Singh, 1993) + 60 mL Sorensen’s phosphate buffer (30 mL KH2PO4 + 30 mL Na2PO4 • H2O, pH 6.9); 2) Leishman’s stain (BDH, England) diluted in a phosphate buffer at pH 6.8 for 30 to 45 min. For silver nitrate staining, the slides were immersed in 2× SSC (0.3 M NaCl, 0.3 M
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BREEDING, CULTIVARS, ROOTSTOCKS, & GERMPLASM RESOURCES Na citrate solution, pH 7.0) for 10 min at room temperature, rinsed briefly in distilled water and air-dried. Two drops of a 100% silver nitrate aqueous solution were placed over the cells. A plastic coverslip was used to spread the stain evenly over the slide. Slides were incubated for 2 to 4 min at 60 °C in a humid chamber. The slides were rinsed a few times in distilled water and air-dried. The slides were dipped in xylene for 30 min and made permanent by mounting in DPX. The slides were examined in a Leitz Diaplan light microscope. Slides with well spread chromosomes were photographed in Ilford PAN F 50 film using a 100 × 1.35 oil immersion objective. Results and Discussion Our improved protocol made it possible to observe all the stages of meiosis in Musa,
including for the first time, the critical prophase stages. This is the first study that shows pachytene chromosomes (Fig. 1A) and the mechanism of crossing-over (Fig. 1B) in Musa. In the past, meiotic studies in Musa focused mainly on the chromosome behavior at metaphase and anaphase (Agarwal, 1987, 1988a, 1988b; Cheesman, 1932a, 1932b; Wilson, 1946a, 1946b, 1946c). The chromosomes at these stages are in their highest level of coiling, appear sharply defined and are stained easily (Fig. 1C). On the contrary, prophase chromosomes are not fully contracted and do not stain well, especially in Musa. Consequently, there have been no published reports on prophase chromosomes of Musa. The pachytene stage of meiosis is preferred for studying chromosome morphology, especially in plants with small chromosomes. Since Musa chromosomes are only 1 to 2 µm in length at
mitotic metaphase (Dolezel et al., 1998; Ortiz, 2000), pachytene analysis may be more suitable for karyotype studies in Musa. Also, pachytene chromosomes provide other landmarks that render them suitable for development of karyotypes. These morphological criteria include the visibility of the centromeres, chromomeres, telomeres, knobs, nucleoli, and the ability to distinguish between euchromatin and heterochromatin (Schulz-Schaeffer, 1980). Meiotic prophase configurations also provide information on structural chromosome changes. For example, Fig. 1A illustrates at least two loop-like structures (arrows) in which the chromosomes are not paired in the pachytene stage in M. acuminata “Calcutta 4’, one of the diploid ancestors of cultivated banana (Simmonds, 1962). ‘Calcutta 4’ has been used as a male parent in banana breeding programs because of its resistance to black
Fig. 1. Silver stained chromosomes of M. acuminata ‘Calcutta 4’ (AA genome), showing (A) pachytene, (B) diakinesis, and (C) anaphase stages. In Fig. 1A, the unpaired chromosome loops (arrows) may represent inversions. Bar = 10 µm.
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Sigatoka, Mycosphaerella fijiensis Morelet, a devastating fungal disease of banana. These loops may represent inversions. Faure et al., (1993) found no structural abnormality in the chromosomes of ‘Calcutta 4’ and reported normal bivalent formation. Although generally considered to be widespread in plants, inversions have been reported (Faure et al., 1993; Shepherd, 1999) but not clearly demonstrated in Musa. This study found that Giemsa and Leishman’s stains were also effective for staining meiotic chromosomes of Musa especially for the well-condensed metaphase and anaphase chromosomes, but silver staining (Pillay and Adeleke, 2001) proved to be most effective for the less contacted prophase chromosomes, including those at pachytene. A thorough knowledge of pachytene chromosome morphology is useful for identifying chromosomes in different genetic stocks, understanding the degree of chromosomal homology, and ascertaining problems of chromosomal aberrations (Jahnavi and Murty, 1985). Well- stained pachytene chromosomes were used to investigate the amount of DNA in euchromatin and heterochromatin in tomato chromosomes (Petersen et al., 1996). The silver staining procedure has formed the basis for similar studies in Musa in our laboratory. The adoption of enzymatic means to digest the rigid cell wall of the microspore mother cells and the use of 3 ethanol : 1 acetic acid to induce bursting of the protoplasts were critical for success of the method reported herein. Since physical pressure was not applied to the cells, the chromosomes retained their original shape and size. Although protoplast techniques have been widely used for mitotic studies, it has been used less frequently for meiotic preparations. Our study showed that it is also advantageous for meiotic studies for observing features such as chiasma formation, the various chromosome associations at diakinesis and anaphase chromosome separation. (Fig. 1 B and C). Since similar methods yielded chromatin that was highly amenable to meiotic FISH (fluorescent in-situ hybridization) in Sorghum bicolor (Zwick et al., 2000), it may be possible to carry out similar studies in Musa. Microspore mother cells in Musa with premeiotic and meiotic stages are not easily extruded from the anthers as generally described for most plants. A cross-section of the anther (data not shown) revealed that the microsporocytes are attached to a central cylinder of cells and closely surrounded by tapetal tissue. Therefore it is necessary to remove the rods of microsporocytes from the anthers and gently
macerate the tissue to release the microspore mother cells. The LB01 buffer, originally used by Dolezel et al. (1997) to isolate nuclei from Musa leaf tissue for flow cytometry, served as a stable medium for the released cells. The centrifugation stages were essential for concentrating the larger microspore mother cells and separating them from the smaller cells that constitute the anther and is also important for removing other debris from the preparations. The absence of suitable cytological techniques for observing meiotic chromosomes in Musa has restricted the development of pachytene karyotypes, cytological characterization of new hybrids, identification of nuclear restitution mechanisms and development of aneuploid stock. Our study provides an improved approach to examine all the meiotic stages in Musa. Literature Cited Agarwal, P.K. 1987. Cytogenetical investigations in Musaceae. II. Meiotic studies in eight male sterile triploid banana varieties of India. Cytologia 52:451–454. Agarwal, P.K. 1988a. Cytogenetical investigations in Musaceae. III. Meiotic studies in diploid Musa species and banana varieties of India. Cytologia 53:359–363. Agarwal, P.K. 1988b. Cytogenetical investigations in Musaceae. IV. Cytomorphology of an interspecific triploid hybrid of Musa acuminata Colla. X M. rubra Wall. Cytologia 53:717–721. Cheesman, E.E. 1932a. Genetic and cytological studies of Musa. I. Certain hybrids of the Gros Michel banana. J. Genet. 26:291–312. Cheesman, E.E. 1932b. Genetic and cytological studies of Musa. II. Hybrids of the Mysore banana. J. Genet. 26:313–316. Cheesman, E.E. and L.N.H. Larter. 1935. Genetic and cytological studies of Musa. III. Chromosome numbers in the Musaceae. J. Genet. 30: 31–50. Cheesman, E.E. and K.S. Dodds. 1942. Genetic and cytological studies of Musa. IV. Certain triploid clones. J. Genet. 43:337–357. Crouch, J.H., D. Vuylsteke, and R. Ortiz. 1998. Perspectives on the application of biotechnology to assist the genetic enhancement of plantain and banana (Musa spp.). Electronic J. Biotechnol. 1. HtmlResAnchor http://www.ejb.org/content/ vol1/issue1/index.html. Dantas, J.L.L., K. Shephard, W. dos S. Soares Filho, Z.J.M. Cordeiro, S. Silva de Oliveira, and A. Silva Souza. 1993. Citogenetica e melhoramento genetico da bananeira (Musa spp.) Documentos CNMPF 48. EMBRAPA, Cruz das Almas, Brazil. Dolezel, J., M.A. Lysak, I. Van den Houwe, M. Dolezelova, and N. Roux. 1997. Use of flow cytometry for rapid ploidy determination in Musa. Infomusa 6:6–9. Dolezel, J., M. Dolezelova, N. Roux, and I. Van den Houwe. 1998. A novel method to prepare slides for high resolution chromosome studies
in Musa spp. Infomusa 7:3–4. Faure. S., F. Bakry, D. Gonzalez De Leon. 1993. Cytogenetic studies of diploid bananas. In: J. Ganry (ed.) Breeding banana and plantain for resistance to diseases and pests. CIRADINIBAP, Montpellier. Jahnavi, M.R. and U.R. Murty. 1985. A preliminary pachytene analysis of two species of Arachis L. Theor. Appl. Genet. 70:157–165. Ortiz, R. 2000. Understanding the Musa genome: An update. In: Craenen K., R. Ortiz, E.B. Karamura, and D.R. Vuylsteke (eds.). Proc. First Intl. Conf. Banana and Plantain for Africa. Acta Hort. 340:157–168. Osuji, J.O., B.E. Okoli, and R. Ortiz. 1996. An improved procedure for mitotic studies of the Eumusa section of the genus Musa L. (Musaceae). Infomusa 5:12–14. Osuji, J.O., G. Harrison, J. Crouch, J.S. HeslopHarrison. 1997. Identification of the genomic constitution of Musa L. genotypes (bananas, plantains and hybrids) using molecular cytogenetics. Ann. Bot. 80: 787–793. Osuji, J.O., G. Harrison, J. Crouch, J.S. HeslopHarrison. 1998. Molecular cytogenetics of Musa species, cultivars and hybrids: Location of 18S5.8S -25S and 5S rDNA and telomere-like sequences. Ann. Bot. 82:243–248. Petersen, D.G., H.J. Price, J.S. Johnston, and S.M. Stack. 1996. DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes. Genome 39:77–82. Pillay, M. and M.T.V. Adeleke. 2001. Silver staining of Musa L. chromosomes. Cytologia 66:33– 37. Pillay, M., A. Tenkouano, and J. Hartman. 2002. Future challenges in Musa breeding. In: M. Kang (ed.). Crop improvement: Challenges in the twenty-first century. Food products Press, New York. Robinson, J.C. 1996. Bananas and plantains. CAB Intl., Wallingford, Oxon, U.K. Schulz-Schaeffer J. 1980. Cytogenetics: Plants, animals, humans. Springer-Verlag, New York. Shepherd, K. 1999. Cytogenetics of the genus Musa. International network for the improvement of banana and plantain, Montpellier, France. Simmonds, N.W. 1962. The evolution of the bananas. Longmans Green and Co., London. Singh, R.J. 1993. Plant cytogenetics. CRC Press. Boca Raton, Fla. Wilson, G.B. 1946a. Cytological studies in the Musae. I. Meiosis in some triploid clones. Genetics 31:241–258. Wilson, G.B. 1946b. Cytological studies in the Musae. II. Meiosis in some diploid clones. Genetics 31:475–482. Wilson, G.B. 1946c. Cytological studies in the Musae. III. Meiosis in some seedling clones. Genetics 31:483–493. Zwick, M.S., M.N. Islam-Faridi, H.B. Zang, G.L. Hodnett, M.I. Gomez, J.S. Kim, H.J. Price and D.M. Stelly. 2000. Distribution and sequence analysis of the centromere-associated repetitive element CEN38 of Sorghum bicolor (Poaceae). Amer. J. Bot. 87:1757–1764.
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