Proe. Indian Acad. Sci., Vol. 87 B, (Plant Sciences-2), No. 5, May 1978, pp. 109-112, ~) printed in India. Stomatal chloroplast number in diploids and polyploids of Gossypium R KRISHNASWAMI and R ANDAL Central Institute for Cotton Research, Regional Station, Coimbatore 641 003 MS received 2 July 1977; in final form 7 November 1977 Abstract. Stomatal chloroplast count technique as a tool to identify the ploidy status was evaluated in species, hybrids and polyploids (natural and induced) involving the A, B and D genomes in Gossypium. The differences between group means were significant. Intra group differences were significant for diploids and hexaploids but this was not due to the genomes involved. Thus some varieties of diploids, G. arboreum (d2) were on par with G. raimondii (Ds). The percentage increase in chloro- plast number per stoma was 25 for triploids, 72 for tetraploids and 102 for hexaploids taking the diploid number as the base. Ploidy per se had great impact on chloroplast number. It appears that stomatal chloroplast count technique can be used as a rapid method to identify polyploids in the genus Gossypium. Keywords. Stomatal chloroplast; Gossypium; polyploids. 1. Introduction Chloroplast-count technique has been used as a method to test the ploidy level in several crop plants. In Trifolium species, Bingham (1968) found that the guard cell chloroplast number effectively distinguished the ploidy level except for diploids versus triploids. He observed that ploidy level per se had a greater influence on chloroplast number in guard cells than does the genome source. Najcervska and Speckmann (1968) studied chloroplasts of different levels of ploidy in red dover, berseem and white dover. They observed an increase in stomatal plastids with the doubling of chromo- some number. Hermsen and De Boer (1971) noticed a significant increase in chloro- plast number in guard cells of diploid and tetraploid species of Solanum. In cotton, stomatal chloroplasts were used to identify haploids and diploids (Chaudhri and Barrow 1975). Cytogenetical investigations are in progress at this institute and many alloploids at 3x, 4x and 6x levels involving different genomes had been synthesised, Using these a study was undertaken to relate the chloroplast number in stomata with ploidy and genome constitution and the results are presented. 2. Materials and methods The genus Gossypium has species with 26 and 52 chromosomes. In the present study all the 4 cultivated species viz. G. herbaceum L., G. arboreum L., G. hirsutum L., and G. barbadense L. were used along with the wild diploid species like G. anomalum Wawr., G. thurberi Tod., G. raimondii Ulb. and G. gossypioides Standl. Polyploids 109 110 R Krishnaswami and R Andal Table 1. Species, hybrids and polyploids of Gossypium used in this study Group Speeies/Polyploid Genome Chromosomenumber (2n) Ploidy I G. herbaceum Ax 26 Diploid G. arboreum A~ 26 ,, G. raimondii D5 26 ,, G. thurberi D1 26 ,, G. gossypioides De 26 ,, II G. hirsutum x G. thurberi (AD)iD1 39 Allotriploid G. hirsutum • G. anomalum (AD)IBi 39 ,, G. barbadense • G. thurberi (AD)2D1 39 ,, HI G. hirsutum (AD)I 52 Tetraploid G. barbadense (AD)2 52 ,, G. hirsutum x G. barbadense F~ (AD)I x (AD)z 52 ,, 2(G. arboreum x G. anomalum) 2(A~Bx) 52 Allotetraploid 2(G. arboreum • G. anomalum) • G. barbadense 2(A2B1) • (AD)I 52 ,, 2(G. arboreum • G. anomalum) • G. hirsutum 2(A2Bx) • (AD)I 52 ,, 2(G. herbaceum x G. arboreum) 2(AxA~) 52 ,, IV 2(G.hirsutum x G. raimondii) 2(ADx • Ds) 78 Allohexaploid 2(G. hirsutum x G. anomalum) 2(AD1 • Bx) 78 ,, 2(G. barbadense • G. thurberi) 2(ADs x D1) 78 ,, 2(G. barbadense • G. gossypioides) 2(ADI x D6) 78 ,, synthesised by crossing cultivated tetraploids with cultivated or wild diploid species were used. The materials used along with their genomic constitution and ploidy status are described in table 1. The cultivated species were represented by 3 or 4 varieties or races but the wild species and synthetic polyploids by one genotype only. Leaves were collected from field or pot grown plants which were exposed to natural light and epidermal peels were taken from the abaxial side. These were stained with potassium iodide-iodine solution and mounted in glycerol. Chloroplasts in the guard cells were counted under a magnification of 675 X. Counts were made from 20 stomata per sample. 3. Results The stomatal chloroplast number of different species, hybrids and polyploids are given in table 2. The material studied was divided into 4 groups on the basis of ploidy. The mean number of chloroplasts per stoma was 11"73 for diploids (group I), 14.67 for allotriploids (group II), 20.24 for tetraploids, natural and synthetic, (group III) and 23"80 for hexaploids (group IV). Statistical analysis of the mean was done within and between the groups. The group mean differences were significant at 0.01 probability level. In the tetraploid group the highly diploidized aUotetraploids like G. hirsutum and G. barbadense were on par with the synthetic tetraploids of different genomic constitutions developed by colchicine technique. There was no significant difference between entries within this group. The hexaploids had signific- ant difference among themselves but as a group, they were distinct from the tetra- ploids. Stomatal chloroplast number in Gossypium ! 11 Table 2. Stomatal chloroplast number in species, hybrids and polyploids of Gossypium Group Species, race or variety hybrid Number of Chloroplasts per stoma combination Range Mean 4- S.E. I G. herbaceum var. africanum 9 -- 14 11"25:t:1"80 G. herbaceum var. V. 797 9 -- 15 11.50-t-2.58 G. herbaceum var. Sujai 10 -- 14 12.504-1-11 G. arboreum var. K 7 9 -- 14 11.654-2.45 ,, var. red 8 -- 13 10.204-2.27 ,, race cernuum 10 -- 14 12.205:1.33 G. raimondii 8 -- 14 10"804-2.59 G. thurberi 11 -- 15 12.604-1.94 G. gossypioides 11 -- 15 12.854-0.97 II G. hirsuturn x G. thurberi 14 -- 17 15-05 4-1"21 G. hirsutum x G. anomalum 13 -- 16 14"004-0.95 G. barbadense x G. thurberi 13 -- 17 14.954-1.51 III G. hirsutum var. MCU. 5 18 -- 22 19"75-4-1"56 ,, var. Reba B-50 19 -- 22 20"404-1-11 ,, var. PRS. 72 18 -- 22 20-004-2.11 ~9 var. Okra 19 -- 22 19'954-0.99 G. barbadense var. Suvin 18 -- 22 20.054-1.31 ,, var. Pima S. 1 19 -- 23 20.504-1.53 ,, var. Sujata 19 -- 22 20.305:1"72 G. hirsutum • G. barbadense F1 19 -- 22 20"154-1.92 2(G. arboreum • G. amomalum) 19 -- 24 20"90-t-1"99 2(G. arboreum • G. anomalum) • G. hirsutum 18 -- 23 19"904-1-78 2(G. arboreum • G. anomalum) x G. barbadense 18 -- 23 20.154-1.92 2(G. herbaceum • G. arboreum) 19 -- 24 20"854-2.11 IV 2(G. hirsutum x G. raimondii) 22 -- 26 23"40:t: 1"41 2(G, hirsutum x G. anomalum) 20 -- 26 22"55_-1-2.32 2(G. barbadense x G. thurberi) 22 -- 28 24.85:t:2.97 2(G. barbadense x G. gossypioides) 22 -- 28 24"404-2.67 The percentage increase in chloroplast number per stoma was 25 for triploids, 72 for tetraploids and 102 for hexaploids taking the diploid number as the base. Intra group differences were not significant for triploids and tetraploids but were significant in diploids and hexaploids. In diploids, there was no significant difference in mean number of chloroplasts per stoma between A and D genomes. Thus G. herbaceum var. africanum, var V. 797, G. arboreum var. K. 7, red arboreum and G. raimondii were not significantly different. Hexaploids involving G. hirsutum as one of the parents had lower number of chloroplasts compared to hexaploids with G. barbadense as a parent. 4. Discussion A technique for rapid identification of haploids as well as polyploids will be of considerable use in cytogenetical investigations where a large number of plants have -~ to be assessed for their ploidy status during a season. Chromosome counting .in somatic tissue or during meiosis is a time consuming lrrOceSS and requires skilled- assistance. A rapid initial screening method with a fair amount of accuracy will minimise the labour involved in routine chromosome counts. The number of P. (B).--3 112 R Krishnaswami and R Andal chloroplasts in guard cells of stomata had been used as an index of ploidy in many crops. Chaudhri and Barrow (1975) working on cotton found that haploid plants of G. hirsutum and G. barbadense had a mean number of 10.46 chloroplasts in their stoma as against 21.00 in the diploids. The ratios of chloroplasts in haploids to diploids was 1:2, the same ratios as their chromosome numbers. They concluded that results of chloroplasts count correspond with those of root tip count of chromosomes. In the present study, we have applied this technique to a large number of polyploids of diverse genome combinations. Irrespective of the genome or genomes involved, the chloroplast number of stoma was related to ploidy. Thus ploidy level per se had a greater influence on chloroplast number than does the genomic constitution. This finding is similar to that of Bingham (1968) in alfalfa. Long established natural tetraploids and new, synthetic tetraploids produced by colchicine treatment had similar chloroplast number. Among the diploid species studied there were significant differences between entries for mean number of chloroplasts per stoma but this was not due to the genome difference. The cultivated diploid species were represented by many genotypes but the wild species had limited representation.