CYTOGENETIC STUDIES IN THE GENUS PERSEA (LAURACEAE).

I. KARYOLOGY OF SEVEN SPECIES1

ARMANDO GARCIA Ve2 Rama de Genetics, Colegio de Postgraduados,

Escuela Nacional de Agricultura, S.A .G., Chapingo, Mexico, Mexico

Chromosome number determinations were made on 137 collections s f seven Persea species. Persea americana Mill., P . schiedeana Nees, and P . aff. cinerascens Rands, had a diploid number of 24 as reported earlier. The first chromosome counts for four species are reported. Persea hintonii Allen (2n =48) is the first tetraplsid species found in this genus. Persea indica (L.) Spreng. (2n=24) is the first Old World species to have its chromosome number reported. Persea donnell-smithii Mez and P . pachypoda Nees d so have the diploid number (2n-24). One triploid (2n=36) and one tetraploid (2n =48) individual were found in P. americana. Based on stomata1 size, their maternal plants were considered to be triploid and diploid, respectively.

This is the first karyotype study in Persea and also in the family Eauraceae. The karyotype in Persea is asymmetric. The chromosomes range in size from 2.3 pm to 6.1 pm. Persea americana has one pair of satellited chrsmosomes, which is the largest pair, two metacentric pairs and nine submetacentric pairs. Two of the submetacentric pairs are highly heterochromatic and both are attached to the nucleolus. A11 the other species have karystypes very similar to P. americana .

Introduction The family Lauraceae includes the genus Persea and about 40 other genera which

are mainly tropical plants distributed in the New World (Koop, 1966; Hutchin~on, 1969). Some Persea species such as P . arnericana Mill. and P. schiedeana Nees are economically important because of their buttery fleshed fruits known commonly as avocado fruits (Koop, 1966; Garcia, 1972). Some others are also important in avocado breeding as sources of tolerance to root rot disease caused by Phytophthora cinnurnorni Rands (Galindo and Zentmyer, 1966).

Many wild species grow in Mexico, and have been collected by several workers and used in avocado breeding programs (Schroeder, 1951; Zentmyer and Schroeder, 1954, 1955; Zentrnyer, 1952, 1953, 1961; Bringhurst, 1954; Frolich et a l . , 1958; Schroeder and Zentmyer, 1958; Bergh, 196 1 ; Galindo and Zentmyer, 1966). Persea americana, the most economically important species in this genus, also grows widely in Mexico from the Pacific coast to the Gulf of Mexico, from southern to northeastern states, and from low to high elevations. This species grows commonly in the states of Nayxit, Sindoa, Michoacm, Mexico, Morelos, Puebla, Veracruz, Guerrero, Oaxaca, Chiapas, Tabasco, Yacatan, San Luis Potosi, Nuevo Leon and Tamaulipas; and also but less frequently in Guanajuato, Queretaro and Hidalgo. In some areas, P . americana grows together with wild species, for example, with P . aff. cinerascens (examined taxonomically, but not identified with full certainty; morphologically, however, it is closest to P . cinerascens Blake, and is expressed as P . aff. cinerascens). Intermediate forms between P . arnericana and P . aff. cinerascens grow in Michoacan and are named as "aguacate blanco" (' 'white avocado"). In Mexico, P . arnericana grows with P . aff.

'Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan, No. 383. The work was supported by the Overseas Technical Cooperation Agency (OTCA) of Japan and Consejo Nacional de Ciencia y Tecnologia (CONACYT) of Mexico. ?resent address: Laboratory sf Genetics, Faculty of Agriculture, Kyoto University. Kyoto 606, Japan. Manuscript received January 3, 1935.

Can. J. Genet. Cytol. 17: 173-180, 1975.

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cipoerascens and P . hipoto~~ii. In Veracruz, P , americana grows together with P . schiedeana, a semi-cultivated species, and some intermediate forms between them are observed. Usually Persea species are propagated sexually and they, especially P . americana, show great variation in fruit shape, size, color and quality as reported by Anderson ( 1 950).

Some cytslogical information has previously been published on Persea species. The chromosome number of 2a=24 has been reported for some species by several authors; i. e., for P . americana (Bawden, 1940, 1945; Schroeder, 1952), P . arnericanw. var. nubigena (Will.) Koop (Bringhurst, 1954, under the name of P . nubigena), P . aff. cinerascens (Garcia, 19781, P . palustris (Raf.) Sarg. (Bowden, 1945), P . borbsnia (L.) Spreng., P . kongipes (Schlecht.) Meissn., P . floccssa Mez (Bringhurst, 1954), and P . schiedeana Nees (Garcia, 1972).

This paper reports the chromosome number and chromosome morphology (karyotypes) from studies made on 137 collections consisting of seven Persea species, most of which were collected by the author from many different places in Mexico.

Materids and Methods The species examined were P . americana Mill., P . schiedeana Nees, P . aff.

cinerascens (see above), P . indica (L.) Spreng . , P . hintonii Allen, P . donnelk-smithii Mez and P . pachypoda Nees. As many as 120 different strains of P . americana were collected from many different locations in 1 1 states (Michoacan, Guanajuato, Quere- taro, Morelos, Mexico, Hidalgo, heb la , Veracmz, Oaxaca, Tabasco and Yucatan). The collections showed variations in size, shape and color of fruit. For P . schiedeana, strains were collected from Veracruz. Collections for P . aff. cinerascens were examined from Michoacan and Mexico, and for P . hintsnii from Tejupilco and Tenerias, in Mexico, but only one collection each for P. indica, (the only Old World species examined), P , donnell-smithii and P . pachypsda were examined (obtained from Dr. George A. Zentmyer, University of California at Riverside, U. S .A. ).

The seeds were germinated in a greenhouse in Chapingo. The seeds of P . hintonii, however did not geminate in the greenhouse, so germinating seeds were sought at both habitats, but could be collected only from the humus along the riverside at Tenerias. Root tips of about 30 mm in length were collected and used for cytological observation. To observe the morphology of Persea chromosomes, the technique of Garcia (1972) was modified by combining suitable pretreatment and critical staining with phase- contrast microscopy. Growing root tips were pretreated in cold water at about 0°C (Baily, 1954) for 24 h, fixed in Farmer's fixative for 48-72 h, and stored in a refrigerator after replacing the fixative with fresh solution. The root tips were hydrolyzed in 1N HCB at 60'C for 45-60 min and stained in aceto-carmine overnight, the staining k i n g accelerated by boiling the root tips for 10-28 sec in aceto-carmine. A squash preparation of a very small portion of the meristem placed in a drop of 45% acetic acid was made using a blunt needle point and pressing enough to spread the chromosomes in the cytoplasm. In some cases, about 3 h treatment at room temperature with cytase taken from snails was also performed after staining, to make the tissue softer. For observing the chromosomes, a phase-contrast microscope was used.

For karyotype study, chromosomes were drawn and measurements were obtained using an ocular micrometer with x 1506 magnification. At least three metaphase cells for each seedling were observed.

Herbarium specimens were made for all the materials studied and have been deposited in the Rama de Genetica, Colegio de Postgraduados in Chapingo, Mexico. Two seedlings from each collection of this study are growing in the greenhouse of the same Institution.

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KARYOLOGY OF PBRSEA SPECIES

Results Chromosome Number Determinations

Persea americana Mill.: Four or five seedlings were examined cytologically for each of 120 strains collected and a somatic chromosome number of 2n =24 was found in all of them (Fig. IA), except for two individuals of two different strains but from the same place, San Juan de la Vega, Guanajuato. One of the exceptional seedlings was found to have 2n -36 chromosomes and the other 2n =48 (Fig. 1B). The morphological characteristics of the triploid seedling were almost normal, whereas the tetraploid seedling was vigorous and had a darker green color and bigger cells (about double-sized stomatal cells) as compared with diploids in the same strain. In an attempt to identify their origins, some mature fresh leaves were collected from their maternal trees, and the size of stomatal cells was examined. The maternal tree of the tetraploid seedling was judged to be diploid because of the normal stomatal cell size, whereas that of the triploid seedling was considered to be triploid since the stomatal cells of the maternal tree were of intermediate size between diploid and tetraploid. The fact that the fruits set on this maternal tree of the triploid seedling have much smaller seeds on the average as compared with diploids (16.6% weight ratio of seedlfruit in this case vs. 32.3% in diploids) (personal communication from the Bepartamento de Fruticultura, Instituto Nacional de Investigaciones Agricolas, S. A. G., Mexico) seems to support the triploid nature of this maternal tree. The occurrence of such polyploids in this economically important species may be useful in the breeding of avocado, for vigor, smaller seeds, or possible tolerance to Phytophthora cinnamomi.

Persea schiedeana Nees: All of 40 seedlings of ten different strains from Veracruz were found to have 2n =24 chromosomes; this chromosome count agrees with an earlier report (Garcia, 1972). Persea schiedeana, the species of second economic importance in Mexico and some Central American countries, belongs to the americana group. It is thought to be the closest to P. americana var. nubigena (Koop, 1966). Some intermediate types between this species and P. americana were observed in Veracruz, supporting such a close relationship.

Persea aff. cinerascens (see above): Diploid chromosome number of 2n -24 was counted in 20 seedlings examined of both collections from Michoacan and Mexico, and is in accordance with the count made for an earlier collection from Michoacan (Garcia, 1 970).

Persea hintonii Allen: Somatic chromosome number of 2n =48 was observed in d l the material (ten germinating seeds) collected. This is the first chromosome count for this species and is the first tetraploid species reported in the genus.

Persea indica (L.) Spreng. : All of 17 seedlings were determined to have 2n =24 chromossmes. This chromosome count is the first for this wild species from the Old World,

Persea donnell-smithii Mez.: The 20 seedlings examined were determined to have 2n =24. This is the first chromosome count reported for this species.

Persea pachypoda Nees: The somatic chromosome number of 2n =24 was counted in all of 20 seedlings, and this is the first chromosome count for this species.

Karyotype The chromosomes of Persea species are small, varying in length from 2.3 to 6.1

pm at metaphase and thus showing an asymmetric karyotype (Levitsky, 1931). The 24 chromosomes of P . americana as shown in Fig. 1A may be classified into four groups by their size. The largest three pairs form the first group, and one of them (No. 1), possesses a secondary constriction in the short arm, whereas the other two pairs (Nos. 2 and 3) do not have satellites. The second group consists of two chromosomes of medium

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KARYOLWY OF PERSEA SPECIES 177

Fig. 2. A. Idiograrn and B. Karyotype of B. americana

size (Nos. 4 and 5) which differ from each other in their arm ratio; the third group includes five chromosomes (Nos. 6, 7, 8, 9 and 10) which have no clear differences among them but are smaller than the second group; the fourth group includes the smallest two chromosomes (Nos. 11 and 121, which are about the same length (Fig. 2). Based on the position of the centromere (Levan et al., 19651, two pairs (Nos. 5 and 1 1 ) are regarded as being metacentrics and the remainder are submetacentrics.

Both arms of chromosome 9 and 10 are highly heterochromatic (Fig. 3) and carry nucleolus organizers.

The karyotypes of other species are almost identical to that of P . americana described above. It was impossible to find any obvious differences in karyotype between species.

Discussion The earliest angiosperms are presumed to have been woody. Consequently,

speculations concerning the basic chromosome number in primitive woody plants and the manner by which the present species arose are of much interest. In the family Lauraceae, x=12 is postulated to be the original chromosome number (Mehra and Bawa, 1969) and, as these authors supposed, this family evolved at the diploid level with minor changes in chromosome morphology.

For the Persea species identified as occurring in the Western Hemisphere (Koop, 1966) the chromosome numbers have been reported for only some of them, namely, P. americana, P. schiedeana, P . aff. cineraseens, P . borbonia, P . longipes and P . floccosa, all of which have been reported as 2n -24. The chromosome numbers reported here support earlier reports for the first three species and agree with reports for the others. The new reports of P. hintonii, the only Old World species reported here, P. pachypoda and P. donnell-smithii are in agreement with the diploid number of 24 chromosomes. An exception to the diploid number of 2n -24 in Persea species reported to date, was observed in P. indica, which was found to have 2n-48, and which was the only tetraploid species reported in this genus. However, tetraploid species have been reported in the Lauraceae, in the genera Litsea, Cinnarnornurn and ~Veolitsea (Mehra and Bawa, 1969).

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KARYOLOGY OF PERSEA SPECIES 179

The presence of some intermediate forms in the genus Persea , and in the whole Persea americana complex, which includes var. americana, var. drymifodia and var. nubigena together with P. schiedeana and P, floccosa, suggests great homology in chromssome structure. Bawa (Mehra and Bawa, 1969), observed that meiosis in putative hybrids between species in the Lauraceae was normal. So, it can be inferred that differentiation of Persea species has been caused by gene mutation. However, detailed pachytene analysis in which minute structural differences could be revealed, if present, must be carried out to verify this hypothesis.

This is the first report on the karyotype for species of Persea and also for any species in the family Lauraceae. A comparison of the karyotypes of the Persea species suggests their chromosome structure is very similar.

Acknowledgments The author wishes to express his sincere gratitude to Dr. George A. Zentmyer,

Professor of Plant Pathology, University of California at Riverside, U.S.A. for providing seeds of some species used in this study. I am also indebted to Dr. Sadao Ichikawa of the Laboratory of Genetics, Kyoto University, Japan, for his criticism of the manuscript and to Dr. Koichiro Tsunewaki, Professor of the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, for accepting me in his laboratory to carry out my doctoral studies and for his criticism in reading the manuscript.

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T. sessile E. Bull. Tort. Bot. Club 81: 414-42 1. Bergh, 0. 1961. Breeding avocado at C. R. C. Yearbook Calif. Avocado Soc. 1361: 68-86. Bowden, W. M. 1940. The chromosome complement and its relationship to cold resistance in

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