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Stomatal Characteristics at Different Ploidy Levels inCoffeaL.
Annals of Botany 80 : 689–692, 1997
SHORT COMMUNICATION
Stomatal Characteristics at Different Ploidy Levels in Coffea L. M. K. M I S H R A Biotechnology Centre, Unit of Central Coffee Research Institute, Coffee Board, Manasagangothri, Mysore-570 006, India Received : 11 April 1996
Accepted : 30 June 1997
Stomatal frequency, epidermal cell frequency, stomatal guard cell length and stomatal index were examined at different ploidy levels in Coffea. In general, stomatal and epidermal cell frequency per unit leaf area decreased while stomatal guard cell length increased with an increase in ploidy. The reduction in stomatal frequency at higher ploidy levels was mainly a result of larger epidermal cells. In the case of C. canephora (cultivar S.274) a significant reduction in stomatal frequency was noticed from diploid to tetraploid level which was due to both larger epidermal cell size and less stomatal differentiation at the tetraploid level. Besides the effect of ploidy on stomatal frequency and guard cell length, genotypic differences in stomatal frequency and stomatal guard cell length were also observed among cultivars of the same ploidy level. Although variation in stomatal frequency among cultivars was found to be associated with the difference in stomatal to epidermal cell ratio, variation in guard cell length was attributed to differential genetic architecture. In the present study a highly significant positive correlation (r ¯ 0±82) between stomatal and epidermal cell frequency and high negative correlations between stomatal frequency and guard cell length (r ¯®0±91) and epidermal cell frequency and stomatal guard cell length (r ¯®0±93) were obtained. The study also indicated that stomatal frequency can be predicted with 83 and 87 % accuracy, respectively, by measuring stomatal guard cell length in coffee. # 1997 Annals of Botany Company Key words : Coffea, ploidy level, stomatal characteristics.
INTRODUCTION Stomatal frequency, guard cell length and stomatal plastid number have often been used as morphological markers for identifying ploidy levels in many plant species viz., alfalfa (Bingham, 1968), Gossypium (Krishnaswamy and Andal, 1978), Dactylis (Santen and Casler, 1986), rye grass (Speckman, Post and Dijkstra, 1965), wheat (Wang et al., 1989) and Bromus inermis (Tan and Dun, 1973 ; Lea, Dunn and Koch, 1977). In the genus Coffea, stomatal frequency was studied at different ploidy levels and a negative correlation was established (Franco, 1939 ; Mishra, Prakash and Sreenivasan, 1991 ; Sreenivasan, Prakash and Mishra, 1992). However, no attempt has yet been made to examine the factors that influence stomatal frequency at different ploidy levels in this genus. There are probably at least two important hereditary and environmental factors which influence stomatal frequency in mature leaves (Fernandez and Muzica, 1973). Since a number of environmental factors such as light, temperature and moisture affect the size of epidermal cells, the number of stomata per unit surface area does not indicate whether environmental factors directly affect the asymetrical mother cell division or whether the variation in number is due to epidermal cell size. In order to quantify the proportion of epidermal cells that develop as stomata irrespective of the cell size, the concept of stomatal index was established (Salisbury, 1927). By using the stomatal index, Salisbury found a highly significant positive correlation between the number of stomata and the 0305-7364}97}11068904 $25.00}0
number of epidermal cells per unit leaf area in the woodland flora of England. Since then the stomatal index has also been used as an additional morphological parameter by many workers. Teare, Peterson and Law (1971) investigated the stomatal features at three different ploidy levels of smooth brome grass (Bromus inermis) and observed that epidermal cell size was the main factor determining stomatal frequency. In the present study, several stomatal features viz., stomatal frequency, epidermal cell frequency, stomatal guard cell length and stomatal index were examined at different ploidy levels of Coffea with the objective of understanding the relationship between ploidy level and stomatal characteristics.
MATERIALS AND METHODS The details of plant material used for the present study are given in Table 1. Ploidy status of all these materials was confirmed (Sreenivasan et al., 1982). For stomatal measurements the first pair of fully expanded leaves were used. A strip of lower epidermis from the middle portion of the leaf was peeled off and mounted in glycerol after staining with safranin. To determine stomatal guard cell length, 25 randomly selected stomata from five leaves per plant were measured microscopically using an ocular micrometer. Similarly, 25 randomly selected microscopic field areas from five leaves were counted per plant to obtain
bo970491
# 1997 Annals of Botany Company
690
Mishra—Coffee Ploidy Leels and Stomata T 1. Stomatal features at different ploidy leels in Coffea
Mean number of stomatal 0±10 mm−#
Mean number of epidermal cells 0±10 mm−#
Stomatal index (mean)
Leaf area served per stoma (¬10−$ mm#) (mean)
Stomatal guard cell length (µm) (mean)
Species}variety
Ploidy level
Origin}source
C. arabica (Kents) C. arabica (S.795) C. arabica Cauvery}Catimor C. canephora (S.274)
Dihaploid (22) Dihaploid (22) Dihaploid (22)
Natural Natural Natural
33±35 29±95 29±35
75±40 74±10 78±00
33±63 32±42 31±50
2±99 3±33 3±40
17±32 20±47 21±31
Diploid (22)
39±65
69±75
37±01
2±52
19±64
Triploid (natural) Triploid (artificial) C. arabica (Kents)
Triploid (33) Triploid (33)
Commercial variety Natural Crossed
25±50 23±85
82±65 69±70
29±04 30±30
3±95 4±21
20±14 21±47
19±40
48±45
32±32
5±19
27±97
C. arabica (S.795)
Tetraploid (44)
15±80
59±00
27±24
6±39
28±47
C. arabica (Cauvery) C. canephora (S.274) tetraploid C. arabica (S.795) C. arabica (S.795) F test C.D. at 5 % C.D. at 1 %
Tetraploid (44)
Commercial variety Commercial variety Commercial variety Colchicine induced Natural Natural — — —
19±40
47±60
32±56
5±16
30±80
20±85
62±00
30±13
4±79
26±81
10±30 9±55 ** 2±46 3±30
36±30 28±00 ** 5±73 7±69
28±28 29±90 ** 4±71 6±32
9±73 10±55 ** 0±68 0±91
35±46 33±45 ** 1±52 2±05
Tetraploid (44)
Tetraploid (44) Hexaploid (66) Octoploid (88) — — —
stomatal and epidermal cell frequency. Stomatal index (SI) was calculated according to the formula of Salisbury (1927) : SI ¯ S}(ES)¬100, where S is the number of stomata per unit leaf area and E is the number of epidermal cells per unit leaf area.
RESULTS AND DISCUSSION The mean stomatal and epidermal cell frequency, stomatal guard cell length, stomatal index and the leaf area served per stoma at different ploidy levels in Coffea were calculated and the data are presented in Table 1. In all cultivars, stomatal and epidermal cell frequency decreased while stomatal guard cell length increased with an increase in ploidy level. However, no significant difference in stomatal frequency could be found between the hexaploid and octoploid levels of the cultivar S.795, although the mean epidermal cell frequency was significantly different. Stomata are associated with many physiological functions of plants and it is possible that any significant reduction in stomatal number below a threshold level may have adverse effects. In the present study, variation in stomatal frequency and guard cell length was also observed among the cultivars of same ploidy level (Table 1). Among dihaploids of arabica, Kents have a higher stomatal frequency with smaller stomata compared to S.795 and Cauvery, although the epidermal cell frequency, stomatal index and leaf area served per stoma did not show any significant differences between these cultivars. Like dihaploids, differences in stomatal frequency and guard cell length was also observed among
the members of tetraploid group, with the tetraploid cultivar S.795 having the lowest stomatal frequency. Besides stomatal frequency, all other stomatal features also differed significantly between S.795 and other arabica tetraploids. A close inspection of the data indicates that in Kents dihaploid, a higher rate of stomatal differentiation, as manifested by the ratio of stomata to epidermal cells, as well as smaller stomatal size, contributed towards its higher stomatal frequency. However, in the case of the S.795 tetraploid, the lower rate of stomatal differentiation appears to be the main factor responsible for its lower stomatal frequency. Genotypic differences in stomatal frequency and guard cell length have been observed in many crop plant species viz., barley (Miskin and Rasmussen, 1970), soyabeans (Chia and Brun, 1975) Triticale (Teare et al., 1971 ; Sapra, Hughes and Sharma, 1975) and the present observation in coffee is consistent with these reports. No significant differences in stomatal characteristics were observed among the two triploids included in the present study except in epidermal cell frequencies. This difference in epidermal cell frequency is due to differences in epidermal cell size. It is noteworthy that the two triploids included in the present study differ in their origin and hence the differences in epidermal cell size may therefore be attributed to their differential genetic architecture. In C. canephora significant differences in stomatal and epidermal cell frequency, guard cell length, stomatal index and leaf area served per stoma were observed between the diploid and tetraploid levels (Table 1). Tetraploid plants have fewer, but larger, stomata compared to their diploid counterparts. The lower stomatal frequency in tetraploids was due to the larger stomatal and epidermal cell size, as
691
Mishra—Coffee Ploidy Leels and Stomata T 2. Correlation coefficients among stomatal features Sl. No. 1 2 3 4 5
Features Stomatal frequency Epidermal cell frequency Stomatal index Leaf area served per stoma Stomatal guard cell length
1 1±000 — — — —
2 0±817** 1±000 — — —
3 0±769** 0±280 1±000 — —
4 ®0±912** ®0±887** ®0±602* 1±000 —
5 ®0±913** ®0±9345** ®0±5089 0±8989** 1±000
** P ! 0±01, * P ! 0±05.
well as reduced stomatal differentiation affected by polyploidy. In the present study, a highly significant positive correlation (r ¯ 0±82) was obtained between stomatal and epidermal cell frequency (Table 2). A similar positive relationship between stomatal and epidermal cell frequency has also been reported in other crops (Salisbury, 1927 ; Heichel, 1971 ; Teare et al., 1971). In addition, like many other crop plant species, viz. brome grass (Tan and Dunn, 1973) and Triticale (Sapra et al., 1975), there is also a significant negative correlation between guard cell length and stomatal and epidermal cell frequency in coffee (Table 2). Based on the correlation analysis, regression equations were calculated to estimate stomatal and epidermal cell frequency based on guard cell length : stomatal frequency ¯ 39±208®0±6037¬guard cell length (1) epidermal cell frequency ¯ 44±895®322¬guard cell length (2) Based on r# values, stomatal frequency and epidermal cell frequency can be predicted with 83 and 87 % accuracy, respectively, by calculating guard cell length. Like many other plant species e.g. alfalfa (Bingham, 1968) Bromus inermis (Tan and Dunn, 1973 ; Lea et al., 1977), and Triticale (Sapra et al., 1975), differences in stomatal frequencies among ploidy levels in coffee were mainly due to the differences in epidermal cell size rather than differences in the ratio of stomatal to epidermal cells. However, the lower stomatal frequency obtained in C. canephora tetraploids compared to diploids, and S.795 tetraploids compared to S.795 dihaploids was due to both larger epidermal cells as well as a reduced rate of stomatal differentiation associated with the higher ploidy level. Although stomatal and epidermal cell frequency decrease and guard cell length increases with an increase in ploidy level, the stomatal index remains more or less stable and is not significantly affected by polyploidy. However, in case of C. canephora the stomatal index decreased significantly with the increase in ploidy level (Table 1). Stomata are associated with many physiological functions in plants. Carbon dioxide and water vapour resistance are directly related to stomatal index and size (Lea et al., 1977). Since environmental factors influence stomatal frequency, but do not influence stomatal index, the latter could be a more useful morphological parameter for screening cultivars for improved water relations and gas exchange capacities in
plant breeding programmes. Furthermore, in Coffea canephora where pronounced differences in stomatal characteristics were observed among ploidy levels, the stomatal count could be used reliably to screen large plant populations treated with colchicine under polyploid breeding programmes. A C K N O W L E D G E M E N TS The author wishes to thank Professor S. S. Bir and Dr C. S. Srinivasan for offering valuable suggestions and to Dr R. Naidu, Director of Research, CCRI for facilities and encouragement. LITRERATURE CITED Bingham ET. 1968. Stomatal chloroplast in alfalfa at four ploidy levels. Crop Science 8 : 509–511. Chia AJ, Brun WA. 1975. Stomatal size and frequency in soyabeans. Crop Science 15 : 309–313. Couturon E. 1986. Obtention d’haploides spontanes de Coffea canephora pieree par l utilisation du greffage d’embryonos. Cafe Cacao The 3 : 155–160. Fernandez AO, Muzica B. 1973. Effects of some environmental factors on the differentiation of stomata in Spirodela intermedia W. Koch. Botanical Gazette 134 : 117–121. Franco CM. 1939. Relation between chromosome number and stomata in Coffea. Botanical Gazette 100 : 817–827. Heichel GH. 1971. Genetic control of epidermal cell and epidermal frequency in maize. Crop Science 11 : 830–832. Krishnaswami R, Andal R. 1978. Stomatal chloroplast number in diploids and polyploids of Gossypium. Proceedings of the Indian Academy of Science 87B (Plant Science) 109–112. Lea HZ, Dunn GM, Koch DW. 1977. Stomatal diffusion resistance in three ploidy levels of smooth bromegrass. Crop Science 17 : 91–93. Mishra MK, Prakash NS, Sreenivasan MS. 1991. Relationship of stomatal length and frequency to ploidy level in Coffea L. Journal of Coffee Research 21 : 32–41. Mishkin KE, Rasmussen DC. 1970. Frequency and distribution of stomata in barley. Crop Science 10 : 575–578. Salisbury EJ. 1927. On the causes and ecological significance of stomatal frequency with special reference to the woodland flora. Philosophical Transactions of the Royal Society, B 216 : 1–65. Santen EV, Casler EV. 1986. Evaluation of indirect ploidy indicators in Dactylis C. Subspecies. Crop Science 26 : 848–852. Sapra VT, Hughes JL, Sharma GC. 1975. Frequency, size and distribution of stomata in triticale leaves. Crop Science 15 : 356–358. Speckman G, Post Jr JJ, Dijkstra H. 1965. The length of stomata as an indicator for polyploidy in rye grasses. Euphytica 14 : 225–230. Sreenivasan MS, Ramachandran M, Sundar KR. 1982. Frequency of polyploids in Coffea arabica. Proceedings of PLACROSYM IV on Genetics, Plant Breeding and Horticulture. Kasargod, Indian Society for Plantation Crops, 23–28.
692
Mishra—Coffee Ploidy Leels and Stomata
Sreenivasan MS, Prakash NS, Mishra MK. 1992. Evaluation of some indirect ploidy indicators in Coffea L. Cafe Cacao The XXXVI : 199–205. Tan GY, Dunn GM. 1973. Relationship of stomatal length and frequency and pollen grain diameter to ploidy level in Bromus inermis leyss. Crop Science 13 : 332–334.
Teare ID, Peterson CJ, Law AG. 1971. Size and frequency of leaf stomata in cultivars of Triticum aestium and other Triticum species. Crop Science 11 : 496–498. Wang PY, Chen R, Fang R, Wu LP, Zhu Z. 1989. Study of identification of ploidy in pollen plants and wheat using guard cells. Acta Agricultural Uniersities Pekinensis 15 : 141–145.
SHORT COMMUNICATION
Stomatal Characteristics at Different Ploidy Levels in Coffea L. M. K. M I S H R A Biotechnology Centre, Unit of Central Coffee Research Institute, Coffee Board, Manasagangothri, Mysore-570 006, India Received : 11 April 1996
Accepted : 30 June 1997
Stomatal frequency, epidermal cell frequency, stomatal guard cell length and stomatal index were examined at different ploidy levels in Coffea. In general, stomatal and epidermal cell frequency per unit leaf area decreased while stomatal guard cell length increased with an increase in ploidy. The reduction in stomatal frequency at higher ploidy levels was mainly a result of larger epidermal cells. In the case of C. canephora (cultivar S.274) a significant reduction in stomatal frequency was noticed from diploid to tetraploid level which was due to both larger epidermal cell size and less stomatal differentiation at the tetraploid level. Besides the effect of ploidy on stomatal frequency and guard cell length, genotypic differences in stomatal frequency and stomatal guard cell length were also observed among cultivars of the same ploidy level. Although variation in stomatal frequency among cultivars was found to be associated with the difference in stomatal to epidermal cell ratio, variation in guard cell length was attributed to differential genetic architecture. In the present study a highly significant positive correlation (r ¯ 0±82) between stomatal and epidermal cell frequency and high negative correlations between stomatal frequency and guard cell length (r ¯®0±91) and epidermal cell frequency and stomatal guard cell length (r ¯®0±93) were obtained. The study also indicated that stomatal frequency can be predicted with 83 and 87 % accuracy, respectively, by measuring stomatal guard cell length in coffee. # 1997 Annals of Botany Company Key words : Coffea, ploidy level, stomatal characteristics.
INTRODUCTION Stomatal frequency, guard cell length and stomatal plastid number have often been used as morphological markers for identifying ploidy levels in many plant species viz., alfalfa (Bingham, 1968), Gossypium (Krishnaswamy and Andal, 1978), Dactylis (Santen and Casler, 1986), rye grass (Speckman, Post and Dijkstra, 1965), wheat (Wang et al., 1989) and Bromus inermis (Tan and Dun, 1973 ; Lea, Dunn and Koch, 1977). In the genus Coffea, stomatal frequency was studied at different ploidy levels and a negative correlation was established (Franco, 1939 ; Mishra, Prakash and Sreenivasan, 1991 ; Sreenivasan, Prakash and Mishra, 1992). However, no attempt has yet been made to examine the factors that influence stomatal frequency at different ploidy levels in this genus. There are probably at least two important hereditary and environmental factors which influence stomatal frequency in mature leaves (Fernandez and Muzica, 1973). Since a number of environmental factors such as light, temperature and moisture affect the size of epidermal cells, the number of stomata per unit surface area does not indicate whether environmental factors directly affect the asymetrical mother cell division or whether the variation in number is due to epidermal cell size. In order to quantify the proportion of epidermal cells that develop as stomata irrespective of the cell size, the concept of stomatal index was established (Salisbury, 1927). By using the stomatal index, Salisbury found a highly significant positive correlation between the number of stomata and the 0305-7364}97}11068904 $25.00}0
number of epidermal cells per unit leaf area in the woodland flora of England. Since then the stomatal index has also been used as an additional morphological parameter by many workers. Teare, Peterson and Law (1971) investigated the stomatal features at three different ploidy levels of smooth brome grass (Bromus inermis) and observed that epidermal cell size was the main factor determining stomatal frequency. In the present study, several stomatal features viz., stomatal frequency, epidermal cell frequency, stomatal guard cell length and stomatal index were examined at different ploidy levels of Coffea with the objective of understanding the relationship between ploidy level and stomatal characteristics.
MATERIALS AND METHODS The details of plant material used for the present study are given in Table 1. Ploidy status of all these materials was confirmed (Sreenivasan et al., 1982). For stomatal measurements the first pair of fully expanded leaves were used. A strip of lower epidermis from the middle portion of the leaf was peeled off and mounted in glycerol after staining with safranin. To determine stomatal guard cell length, 25 randomly selected stomata from five leaves per plant were measured microscopically using an ocular micrometer. Similarly, 25 randomly selected microscopic field areas from five leaves were counted per plant to obtain
bo970491
# 1997 Annals of Botany Company
690
Mishra—Coffee Ploidy Leels and Stomata T 1. Stomatal features at different ploidy leels in Coffea
Mean number of stomatal 0±10 mm−#
Mean number of epidermal cells 0±10 mm−#
Stomatal index (mean)
Leaf area served per stoma (¬10−$ mm#) (mean)
Stomatal guard cell length (µm) (mean)
Species}variety
Ploidy level
Origin}source
C. arabica (Kents) C. arabica (S.795) C. arabica Cauvery}Catimor C. canephora (S.274)
Dihaploid (22) Dihaploid (22) Dihaploid (22)
Natural Natural Natural
33±35 29±95 29±35
75±40 74±10 78±00
33±63 32±42 31±50
2±99 3±33 3±40
17±32 20±47 21±31
Diploid (22)
39±65
69±75
37±01
2±52
19±64
Triploid (natural) Triploid (artificial) C. arabica (Kents)
Triploid (33) Triploid (33)
Commercial variety Natural Crossed
25±50 23±85
82±65 69±70
29±04 30±30
3±95 4±21
20±14 21±47
19±40
48±45
32±32
5±19
27±97
C. arabica (S.795)
Tetraploid (44)
15±80
59±00
27±24
6±39
28±47
C. arabica (Cauvery) C. canephora (S.274) tetraploid C. arabica (S.795) C. arabica (S.795) F test C.D. at 5 % C.D. at 1 %
Tetraploid (44)
Commercial variety Commercial variety Commercial variety Colchicine induced Natural Natural — — —
19±40
47±60
32±56
5±16
30±80
20±85
62±00
30±13
4±79
26±81
10±30 9±55 ** 2±46 3±30
36±30 28±00 ** 5±73 7±69
28±28 29±90 ** 4±71 6±32
9±73 10±55 ** 0±68 0±91
35±46 33±45 ** 1±52 2±05
Tetraploid (44)
Tetraploid (44) Hexaploid (66) Octoploid (88) — — —
stomatal and epidermal cell frequency. Stomatal index (SI) was calculated according to the formula of Salisbury (1927) : SI ¯ S}(ES)¬100, where S is the number of stomata per unit leaf area and E is the number of epidermal cells per unit leaf area.
RESULTS AND DISCUSSION The mean stomatal and epidermal cell frequency, stomatal guard cell length, stomatal index and the leaf area served per stoma at different ploidy levels in Coffea were calculated and the data are presented in Table 1. In all cultivars, stomatal and epidermal cell frequency decreased while stomatal guard cell length increased with an increase in ploidy level. However, no significant difference in stomatal frequency could be found between the hexaploid and octoploid levels of the cultivar S.795, although the mean epidermal cell frequency was significantly different. Stomata are associated with many physiological functions of plants and it is possible that any significant reduction in stomatal number below a threshold level may have adverse effects. In the present study, variation in stomatal frequency and guard cell length was also observed among the cultivars of same ploidy level (Table 1). Among dihaploids of arabica, Kents have a higher stomatal frequency with smaller stomata compared to S.795 and Cauvery, although the epidermal cell frequency, stomatal index and leaf area served per stoma did not show any significant differences between these cultivars. Like dihaploids, differences in stomatal frequency and guard cell length was also observed among
the members of tetraploid group, with the tetraploid cultivar S.795 having the lowest stomatal frequency. Besides stomatal frequency, all other stomatal features also differed significantly between S.795 and other arabica tetraploids. A close inspection of the data indicates that in Kents dihaploid, a higher rate of stomatal differentiation, as manifested by the ratio of stomata to epidermal cells, as well as smaller stomatal size, contributed towards its higher stomatal frequency. However, in the case of the S.795 tetraploid, the lower rate of stomatal differentiation appears to be the main factor responsible for its lower stomatal frequency. Genotypic differences in stomatal frequency and guard cell length have been observed in many crop plant species viz., barley (Miskin and Rasmussen, 1970), soyabeans (Chia and Brun, 1975) Triticale (Teare et al., 1971 ; Sapra, Hughes and Sharma, 1975) and the present observation in coffee is consistent with these reports. No significant differences in stomatal characteristics were observed among the two triploids included in the present study except in epidermal cell frequencies. This difference in epidermal cell frequency is due to differences in epidermal cell size. It is noteworthy that the two triploids included in the present study differ in their origin and hence the differences in epidermal cell size may therefore be attributed to their differential genetic architecture. In C. canephora significant differences in stomatal and epidermal cell frequency, guard cell length, stomatal index and leaf area served per stoma were observed between the diploid and tetraploid levels (Table 1). Tetraploid plants have fewer, but larger, stomata compared to their diploid counterparts. The lower stomatal frequency in tetraploids was due to the larger stomatal and epidermal cell size, as
691
Mishra—Coffee Ploidy Leels and Stomata T 2. Correlation coefficients among stomatal features Sl. No. 1 2 3 4 5
Features Stomatal frequency Epidermal cell frequency Stomatal index Leaf area served per stoma Stomatal guard cell length
1 1±000 — — — —
2 0±817** 1±000 — — —
3 0±769** 0±280 1±000 — —
4 ®0±912** ®0±887** ®0±602* 1±000 —
5 ®0±913** ®0±9345** ®0±5089 0±8989** 1±000
** P ! 0±01, * P ! 0±05.
well as reduced stomatal differentiation affected by polyploidy. In the present study, a highly significant positive correlation (r ¯ 0±82) was obtained between stomatal and epidermal cell frequency (Table 2). A similar positive relationship between stomatal and epidermal cell frequency has also been reported in other crops (Salisbury, 1927 ; Heichel, 1971 ; Teare et al., 1971). In addition, like many other crop plant species, viz. brome grass (Tan and Dunn, 1973) and Triticale (Sapra et al., 1975), there is also a significant negative correlation between guard cell length and stomatal and epidermal cell frequency in coffee (Table 2). Based on the correlation analysis, regression equations were calculated to estimate stomatal and epidermal cell frequency based on guard cell length : stomatal frequency ¯ 39±208®0±6037¬guard cell length (1) epidermal cell frequency ¯ 44±895®322¬guard cell length (2) Based on r# values, stomatal frequency and epidermal cell frequency can be predicted with 83 and 87 % accuracy, respectively, by calculating guard cell length. Like many other plant species e.g. alfalfa (Bingham, 1968) Bromus inermis (Tan and Dunn, 1973 ; Lea et al., 1977), and Triticale (Sapra et al., 1975), differences in stomatal frequencies among ploidy levels in coffee were mainly due to the differences in epidermal cell size rather than differences in the ratio of stomatal to epidermal cells. However, the lower stomatal frequency obtained in C. canephora tetraploids compared to diploids, and S.795 tetraploids compared to S.795 dihaploids was due to both larger epidermal cells as well as a reduced rate of stomatal differentiation associated with the higher ploidy level. Although stomatal and epidermal cell frequency decrease and guard cell length increases with an increase in ploidy level, the stomatal index remains more or less stable and is not significantly affected by polyploidy. However, in case of C. canephora the stomatal index decreased significantly with the increase in ploidy level (Table 1). Stomata are associated with many physiological functions in plants. Carbon dioxide and water vapour resistance are directly related to stomatal index and size (Lea et al., 1977). Since environmental factors influence stomatal frequency, but do not influence stomatal index, the latter could be a more useful morphological parameter for screening cultivars for improved water relations and gas exchange capacities in
plant breeding programmes. Furthermore, in Coffea canephora where pronounced differences in stomatal characteristics were observed among ploidy levels, the stomatal count could be used reliably to screen large plant populations treated with colchicine under polyploid breeding programmes. A C K N O W L E D G E M E N TS The author wishes to thank Professor S. S. Bir and Dr C. S. Srinivasan for offering valuable suggestions and to Dr R. Naidu, Director of Research, CCRI for facilities and encouragement. LITRERATURE CITED Bingham ET. 1968. Stomatal chloroplast in alfalfa at four ploidy levels. Crop Science 8 : 509–511. Chia AJ, Brun WA. 1975. Stomatal size and frequency in soyabeans. Crop Science 15 : 309–313. Couturon E. 1986. Obtention d’haploides spontanes de Coffea canephora pieree par l utilisation du greffage d’embryonos. Cafe Cacao The 3 : 155–160. Fernandez AO, Muzica B. 1973. Effects of some environmental factors on the differentiation of stomata in Spirodela intermedia W. Koch. Botanical Gazette 134 : 117–121. Franco CM. 1939. Relation between chromosome number and stomata in Coffea. Botanical Gazette 100 : 817–827. Heichel GH. 1971. Genetic control of epidermal cell and epidermal frequency in maize. Crop Science 11 : 830–832. Krishnaswami R, Andal R. 1978. Stomatal chloroplast number in diploids and polyploids of Gossypium. Proceedings of the Indian Academy of Science 87B (Plant Science) 109–112. Lea HZ, Dunn GM, Koch DW. 1977. Stomatal diffusion resistance in three ploidy levels of smooth bromegrass. Crop Science 17 : 91–93. Mishra MK, Prakash NS, Sreenivasan MS. 1991. Relationship of stomatal length and frequency to ploidy level in Coffea L. Journal of Coffee Research 21 : 32–41. Mishkin KE, Rasmussen DC. 1970. Frequency and distribution of stomata in barley. Crop Science 10 : 575–578. Salisbury EJ. 1927. On the causes and ecological significance of stomatal frequency with special reference to the woodland flora. Philosophical Transactions of the Royal Society, B 216 : 1–65. Santen EV, Casler EV. 1986. Evaluation of indirect ploidy indicators in Dactylis C. Subspecies. Crop Science 26 : 848–852. Sapra VT, Hughes JL, Sharma GC. 1975. Frequency, size and distribution of stomata in triticale leaves. Crop Science 15 : 356–358. Speckman G, Post Jr JJ, Dijkstra H. 1965. The length of stomata as an indicator for polyploidy in rye grasses. Euphytica 14 : 225–230. Sreenivasan MS, Ramachandran M, Sundar KR. 1982. Frequency of polyploids in Coffea arabica. Proceedings of PLACROSYM IV on Genetics, Plant Breeding and Horticulture. Kasargod, Indian Society for Plantation Crops, 23–28.
692
Mishra—Coffee Ploidy Leels and Stomata
Sreenivasan MS, Prakash NS, Mishra MK. 1992. Evaluation of some indirect ploidy indicators in Coffea L. Cafe Cacao The XXXVI : 199–205. Tan GY, Dunn GM. 1973. Relationship of stomatal length and frequency and pollen grain diameter to ploidy level in Bromus inermis leyss. Crop Science 13 : 332–334.
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