© 2010 The Japan Mendel Society Cytologia 75(4): 379–387, 2010
Cytomorphological Diversity in Some Species of Impatiens Linn. (Balsaminaceae) from Western Himalayas (India)
Syed Mudassir Jeelani*, Savita Rani, Sanjeev Kumar, Raghbir Chand Gupta and Santosh Kumari
Department of Botany, Punjabi University, Patiala 147 002, India
Received May 28, 2010; accepted August 28, 2010
Summary The genus Impatiens Linn. belongs to the family Balsaminaceae and includes mostly wild as well as commonly cultivated ornamental plants. Nearly 91% of Indian species of Impatiens are reported to be endemic. To generate basic information on genetic diversity required for the improvement of germplasm, the present study has been carried out from the different selected parts of Western Himalayas such as Kashmir (J&K) and the Kangra and Sirmaur districts (H.P). During this study, 23 accessions belonging to 9 species of the genus Impatiens have been cytomorphologi- cally observed. The species being cytologically worked out for the first time on a worldwide basis include 2 species as I.laxiflora (n 7, 8) and I. reidii (n 7). Six aneuploid cytotypes have been reported for the first time for the species I. arguta (n 7), I. bicornuta (n 7), I. brachycentra (n 8), I. glandulifera (n 6), I. scabrida (n 6) and I. sulcata (n 8) on a worldwide basis. The meiotic course in most of these accessions has been observed to be normal except for some of the accessions of I. brachycentra, I. glandulifera, I. scabrida and I. sulcata marked with abnormal meiosis. Out of 4 species (6 accessions) marked with cytomixis, in 2 accessions, one for each of I. scabrida and I. sulcata, the percentage of cytomixis has been seen to be relatively higher with production of heterogenous sized fertile pollen grains. The presence of B-chromosomes in I. balsamina is in conformity with the earlier reports for this species. Morphological comparison at intraspecific level has also been made for the different aneuploid cytotypes of I. brachycentra (n 7 and n 8), I. glandulifera (n 9 and n 6), I. laxiflora (n 7 and n 8) and I. scabrida (n 6 and n 7) highlighting the morphological changes coupled with chromosomal changes.
Key words Cytotypes, Impatiens, Meiotic abnormalities, Western Himalayas.
The genus Impatiens belongs to the family Balsaminaceae and occurs mostly in tropical and subtropical regions of the Old World (Grey-Wilson 1980) with limited number of species present in temperate parts of northern Asia, Europe and north America (Song et al. 2003). It is represented by more than 1000 species on a worldwide basis (Clifton 2000). In India, the concentration of species of Impatiens is well defined viz. tne Western Himalayas (North India), the hills of the north Eastern States and Western Ghats (South India) with a total of 206 species (Vivekananthan et al. 1997), out of which more than 91% are endemic (Chatterjee 1940). The different species of the genus are generally found growing in moist shady places or occasionally as epiphytes in altitudinal range of 800– 3500 m. A perusal of literature shows that significant number of species of Impatiens have been worked out cytologically on a worldwide basis as evident from data compiled for 189 species by Song et al. (2003). The different chromosome numbers reported in the genus are 2n 6, 8, 10, 12, 14, 15, 16, 17,18, 19, 20, 24, 26, 28, 30, 32, 34, 36, 40, 44, 48, 50, 56 and 66 (Song et al. 2003). The different species of the genus Impatiens have medicinal properties such as antioxidant (Shahwar et al. 2010),
* Corresponding author, e-mail: [email protected] 380 S. M. Jeelani et al. Cytologia 75(4) antibacterial and antifungal (Nisar et al. 2010) activities. Ethnobotanically, the different species of the genus are used for the treatment of gonorrhea, external burns and pains in joints (Quereshi et al. 2007). Due to the endemic nature, sympatric distribution (Khoshoo 1957), intraspecific chromosomal variations and introgression among some of the species (Mehra et al. 1968) coupled with the economic importance, cytomorphological studies on different species of the genus Impatiens from relatively less explored areas of the Western Himalayas have been presently undertaken.
Materials and methods For meiotic studies, plant materials were collected from selected localities of different areas of the Western Himalayas. Usual acetocarmine smears of appropriate sized flower buds were made after fixing them in the Carnoy’s fixative. Pollen fertility was estimated by mounting mature pollen grains in glycero–acetocarmine (1 : 1) mixture. Well-filled pollen grains with stained nuclei were taken as apparently fertile, while shrivelled and unstained pollen grains were counted as sterile. Pollen grain size was measured using an occulomicrometre. Photomicrographs of pollen mother cells and pollen grains were made from freshly prepared slides using a Nikon 80i eclipse Digital Imaging System. Voucher specimens are available in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).
Results Detailed cytomorphological studies were carried out on 23 accessions belonging to 9 species of the genus Impatiens. The data regarding locality with altitude, accessions and meiotic chromosome numbers (present and previous) of the presently worked out species has been presented in Table 1. The results for each species with new/additional/varied chromosome counts and abnormal meiotic course are discussed below. I.arguta Hook. f. & Thoms. The single accession of this species from Kangra shows n 7 (Fig. 1). The meiotic course is observed to be normal with high pollen fertility of (94%). I. balsamina L. All the 5 accessions have the same chromosome number n 7 with 1 B-chromosome in the single accession from Boh (Fig. 2).The course of meiosis has been found to be normal in all the accessions with high pollen fertility. I. bicornuta Wall. In Roxb.
The single accession for the species from Sirmaur depicts 7II at metaphase-I (Fig. 3). The meiotic course is found to be normal with almost cent per cent pollen fertility (99%). I. brachycentra Kar & Kir. Out of the 4 different accessions of the species, one each from Bara-gran, Nauradhar and Keller show n 7 (Fig. 4) with normal course of meiosis and high pollen fertility. The accession from Sachapass shows 8II at metaphase-I (Fig. 5) with abnormal meiotic course (Table 2). The cytotype with n 8 shows significant morphological difference from the 1 with n 7 (Table 3). The 3 accessions with n 7 are morphologically identical. I. glandulifera Royle. Both the accessions of the species show different chromosome counts. The accession from
Kangra shows 6II at metaphase-I (Fig. 6) and the meiotic course is found to be normal with high pollen fertility (98%). On the other hand, the accession from Sirmaur exhibits n 9 (Fig. 7) with abnormal meiotic course (Table 2). The 2 cytotypes show some morphological differences (Table 3). 2010 Cytomorphological Diversity in Western Himalayan Impatiens 381
Table 1. Information about locality, altitude with accession numbers and chromosome numbers of presently worked out species of genus Impatiens from the Western Himalayas
Locality/ Chromosome numbers S.No. Taxa Altitide (m)/ Accession numbers Present count *Previous reports
1. I. arguta Hook. Multan, District, n 72n 12, 18, 20 f. & Thoms. Kangra (H.P); 2,300; 52568 2. I. balsamina L. Ranhear, District n 7 Kangra (H.P); 850; 52558 Boh, District n 7 1B 2n 10, 12, 14, 14 2B, 18, Kangra (H.P); 1,900; 20, 24, 26, 28, 28 2B, 52566 56, 56 2B Ganduri, District n 7 Sirmaur (H.P); 2,100; 52535 Nauradhar, District n 7 Sirmaur (H.P); 2,000; 52567 Tral, District n 7 Pulwama (J & K); 2,300; 52508 3. I. bicornuta Haripurdhar, District n 72n 16, 18 Wall. In Roxb. Sirmaur (H.P); 2,300; 52542 4. I. brachycentra Sacchapass, District n 8 Kar & Kir. Kangra (H.P); 3,200; 52559 2n 14 Bara-gran, District n 7 Kangra (H.P); 3,000; 52560 Nauradhar, District n 7 Sirmaur (H.P); 2,000; 52539 Keller, District n 7 Shopian (J & K); 2,350; 52506 5. I. glandulifera Bara-gran, District n 6 Royle. Kangra (H.P); 3,000; 52564 2n 18, 20 Ganduri, District n 9 Sirmaur (H.P); 2,000; 52570 6. I. laxiflora Ganduri, District n 8 No report Edegew. Sirmaur (H.P); 2,000; 52540 Rajgarh, District n 7 Sirmaur (H.P); 1,600; 52538 7. I. reidii Multan, District n 7 No report Kar & Kir. Kangra (H.P); 2,300; 52565 8. I. scabrida DC. Multan, District n 7 Kangra (H.P); 2,300; 52571 2n 14, 16, 18, 20 Boh, District n 6 Kangra (H.P); 1,900; 52563 382 S. M. Jeelani et al. Cytologia 75(4)
Table 1. (Continued)
Locality/ Chromosome numbers S.No. Taxa Altitide (m)/ Accession numbers Present count *Previous reports
8. I. scabrida DC. Haripurdhar, District n 7 Sirmaur (H.P); 2,300; 52541 Ganduri, District n 7 Sirmaur (H.P); 2,000; 52543 Aharbal, District n 6 Kulgam (J & K); 2,250; 52507 9. I. sulcata Wall Aharbal, District n 8 Shopion (J & K); 2,200; 52505 2n 18, 20 Ferozpur, District n 8 Baramulla (J & K); 1,700; 52569
* Previous chromosome number reports are based on literature from Chromosomal Atlases by Fedorov (1969), and Kumar and Subramanium Vol. I (1986); Index to Plant Chromosome Number Reports from 1968 onwards; various Journals, Proceeding volumes and internet.
I. laxiflora Edegew. The 2 accessions from Sirmaur district are found to have different chromosome numbers, n 8 and n 7 (Figs. 8, 9) with normal course of meiosis and high pollen fertility. Some significant morphological variations are noticed between the 2 cytotypes (Table 3). I. reidii Kar & Kir.
The single accession of the species from Kangra shows 7II at metaphase-I (Fig. 10) and the meiotic course is found to be normal with high pollen fertility (96%). I. scabrida DC.
Two accessions from Sirmaur district and one from Mutan are found to have 7II during diakinesis/ metaphase-I where as 2 accessions, one each from the Kangra and Shopion (J & K) districts, are found to have n 6 (Fig. 11). Among these, the accessions with n 7 from Multan and Haripurdhar have some meiotic abnormalities (Table 2) whereas the remaining accessions have normal course of meiosis. The 2 cytotypes show some significant morphological differences (Table 3). I. sulcata Wall.
Two accessions, one each from Aharbal and Ferozpur, are depicting 8II at metaphase-I (Fig. 12) with highly abnormal meiotic course (Table 2).
Discussion Chromosome numbers: There is a lot of confusion regarding the base number of the genus such as x 7, 10 (Khoshoo 1957), x 7 (Jones and Smith 1966; Akiyama et al. 1992), x 8 (Rao et al. 1986) and x 7, 8, 9, 10 (Song et al. 2003). The genus depicts the role of aneuploidy being major evolutionary process as evident from typical dysploid chromosome numbers as 2n 6, 8, 10, 12, 14, 18, 20, 24, 26, 28, 30, 32, 34, 36, 40, 44, 48, 50, 56, 66 and unusual numbers 2n 15, 17, 19 (Song et al. 2003). Any one of or all cytogenetic phenomena, such as irregular segregation, non- 2010 Cytomorphological Diversity in Western Himalayan Impatiens 383
Fig. 1–24. 1-I. arguta, 52568 (2n 14) anaphase-I. 2-I. balsamina, 52566 (n 7 1B) metaphase-I. 3-I. bicornuta, 52542 (n 7) metaphase-I. 4-I. brachycentra, 52560 (2n 14) anaphase-I. 5-I. brachycentra, 52559 (n 8) metaphase-I. 6-I. glandulifera, 52564 (n 6) metaphase-I. 7-I. glandulifera, 52570 (2n 18) anaphase-I. 8-I. laxiflora, 52540 (n 8) diakinesis. 9- I. laxiflora, 52538 (n 7) diakinesis. 10-I. reidii, 52565 (n 7) metaphase-I. 11-I. scabrida, 52563 (n 6) metaphase-I. 12-I. sulcata, 52505 (n 8) metaphase-I. 13-Two PMCs showing cytomixis at telophase-II in I. scabrida, 52571. 14-PMCs showing chromosomal stickiness at metaphase-I in I. sulcata, 52505. 15-PMC showing unoriented bivalent at metaphase-I in I. scabrida, 52571. 16-PMC showing bridge at anaphase-I in I. sulcata, 52505. 17-PMC showing multiple bridges at anaphase-I in I. scabrida, 52571. 18-PMC showing laggards at anaphase-I in I. sulcata, 52569 19-PMC showing laggard at anaphase-I in I. scabrida, 52541. 20-Diad in I. sulcata, 52505. 21- Tetrad with micronuclei in I. scabrida, 52571. 22-Triad with micronuclei in I. sulcata, 52505. 23-Heterogenous sized fertile pollen grains in I. sulcata, 52571 24-Fertile and sterile pollen grains in I. scabrida, 52541. Scale 10 mm. 384 S. M. Jeelani et al. Cytologia 75(4)
Table 3. Data showing morphological comparison between aneuploid cytotypes of I. brachycentra, I. glandulifera, I. laxiflora and I. m) 19.05 18.24 21.95 17.72 17.58 20.98 scabrida m Size ( Cytotype Cytotype S. No. Character (Diploid) (Aneuploid)
Pollen grains Pollen I. brachycentra Chromosome number n 7 n 8 (%) 63.40 30.42 85.0086.30 32.98 31.05 54.68 28.35 82.00 32.73 1. Plant height (cm) 20–30 30–50 Fertility 2. Leaf size (cm) 5.2 2.3 6.1 3.4 3. Flower colour white white-purplish 4. Flower size (cm) 2 3.5 1 1.5 5. Spur size (cm) 0.2 0.5 5.60 6.58 — 4.46 6. Capsule size (cm) 0.5 111.5 10.50 I. glandulifera Chromosome number n 9 n 6
marked with abnormalmarked meiosis from the Western 1. Plant height (cm) 45–55 30–60 2. Leaf size (cm) 5.3 2.2 8.2 2.5 3. Flower size (cm) 3.5 2.5 2 1
Impatiens 4. Spur size (cm) 1 0.5 16.40 12.10 11.54 11.48 10.54 5. Capsule size (cm) 1 3.5 0.4 0.7 I. laxiflora Chromosome number n 7 n 8 1. Plant height (cm) 30–50 30–35 2. Leaf size (cm) 6.4 2.3 6.2 2.5 9.46 — 19.40 14.10 20.40 3. Flower size (cm) 1 0.5 2 0.8 4. Spur size (cm) 0.5 1 5. Capsule size (cm) 0.4 0.6 0.7 1.7 I. scabrida Chromosome number n 7 n 6 Meiotic course showing PMCs with Meiotic course showing 1. Plant height (cm) 40–50 50–60 2.22 4.38 — 6.24 — 2. Leaf size (cm) 10.2 5.3 6.1 2.2 3. Flower size (cm) 2.5 12 1 4. Spur size (cm) 1 0.5 5. Capsule size (cm) 2.5 0.5 1 1.5 — — 9.0 — ** Based on minimum of 10 observations for each value. stickiness bivalents A-I/T-I/ A-I/ T-I/ at T-I/ at M-I (%) at M-I (%) (%) A-II/T-II (%) A-II/T-II (%) T-II Chromosomal Unoriented Bridges at Laggards at Micronuclei disjunction, unequal translocations, centric fusion or centric fission and hybridization, may be involved in the origin of such dysploid A-I M-I
T-I/ T-II T-I/ chromosome numbers (Stebbins 1971). The present chromosome count of n 7 for I.arguta makes an addition to the intra- Cytomixis specific aneuploid cytotypes for the species, which is already reported to exist at diploid 8.670.43 M-II/ T-II 13.58 0.65 0.24 9.49 M-I/M-II
Involved stage
% of PMCs Meiotic levels with 2n 18 (Akiyama et al. 1996) and 2n 20 (Sugawara et al. 1994) on worldwide Himalayas Data on cytomixis, meiotic course, pollen fertility and pollen grain size in different accessions of meiotic course, pollen fertilityData on cytomixis, and pollen grain size in different basis along with another cytotype showing 2n 12 reported from India (Chaterjee and Sharma 1970). The present chromosome Taxa/ Table 2. 52505 52569 52570 52541 52559 0.34 A-I 1.30 — 11.40 16.40 5.02 78.00 32.08 52571
Accessions number, n 7 for I. bicornuta forms a new I. sulcata I. glandulifera I. brachycentra I. scabrda aneuploid report for the species, as it is already 2010 Cytomorphological Diversity in Western Himalayan Impatiens 385 known to exist at diploid level with 2n 18 (Akiyama et al. 1992) on a worldwide basis and 2n 16 (Malla et al. 1978) for Indian cytotype. I. brachycentra showing n 8 adds a new intraspecific aneuploid cytotype to the already existing diploid cytotype, 2n 14 from India (Bhat et al. 1975) as well as from outside India (Khatoon 1991). I.glandulifera showing present chromosome count of n 6 makes a new addition to the existing diploid cytotypes 2n 18 reported from outside India (Dobes et al. 1997) and 2n 20 from India (Chinappa and Gill 1974). I. scabrida exists at the diploid level on worldwide basis as 2n 14 (Akiyama et al. 1992), however diploids with 2n 16 (Sharma and Ghosh 1976), 2n 18 (Chaterjee and Sharma 1970) and 2n 20 (Khoshoo 1955) have been earlier reported from India. It is pertinent to mention here that the presently reported chromosome number n 6 from Kangra and Shopion makes a new aneuploid report for this species on worldwide basis. The present chromosome count (n 8) for I. sulcata is a new additional aneuploid record for the species which was earlier known to exist at diploid level as 2n 18 (Akiyama et al. 1992) on a worldwide basis, with 2n 20 reported from India (Khoshoo 1955). Interestingly, the present chromosome numbers, n 7 and 8 for I. laxiflora make altogether a new chromosomal record of these 2 cytotypes on a worldwide basis. I.reidii with present chromosome count of n 7 is also being cytologically worked out for the first time on a worldwide basis. However, the presence and frequency of B-chromosomes in I. balsamina is in conformity with the earlier reports by Raghuvanshi and Mahajan (1985) known for the species. On the basis of pooled chromosome numbers data, it is obvious that most of the presently investigated species show dysploid numerical chromosome variations viz. I. arguta (2n 14, 18, 20), I. bicornuta (2n 14, 16, 18), I. brachycentra (2n 14, 16), I. glandulifera (2n 12, 18, 20), I. laxiflora (2n 14, 16), I.scabrida (2n 12, 14, 16, 18, 20) and I. sulcata (2n 16, 18). According to data compiled by Song et al. (2003) it is obvious that polyploidy is quite low in this genus with tetraploid level being more common and further, polyploidy is restricted more to south Indian species than north Indian species. Earlier it was thought that the geographical distribution of the species with x 6 and 7 was largely confined to the Indian subcontinent, (Jones and Smith 1966). The species with x 7 and x 10 grow sympatrically in various parts of India (Khoshoo 1957), otherwise species with x 6 and 7 are largely confined to the whole of the Indian subcontinent (Jones and Smith 1966). On the basis of an analysis of massive data, collected by Song et al. (2003) for the genus, he has shown that the chromosome numbers 2n 12, 14, 15, 18, 19, 20, 28 and 36, based on x 6, 7, 9 and 10 are available in the Himalayas (including 2 zones- Western Himalaya and Hills of North Eastern States) against the reports from South India (third zone) with the predominance of species based on x 7 and 10. The comparison is clear as that for both north and south Indian species, the common base numbers remained x 7 and 10 with a preponderance of third base number x 9 being related to north Indian species. It is agreed upon that overall the series of these basic chromosome numbers suggest typical dysploid evolutionary relationships among the species as also shown earlier by Yuan et al. (2004). Meiotic abnormalities: At present the cytomixis is being noted in the different accessions of I. brahycentra, I. glandulifera, I. scabrida and I.sulcata. However, the percentage of PMCs showing cytomixis in all these accessions are low except for the accessions of I. scabrida (52571) and I. sulcata (52505) in which the percentage of cytomixis is moderately high coupled with high chromosomal stickiness (Figs. 13, 14; Table 2). The phenomenon of cytomixis, resulting into transfer of chromatin between PMCs, has already been reported in many Angiospermic plants (Ghanima and Talaat 2003, Ghaffari 2006, Singhal et al. 2008). Besides cytomixis and chromosomal stickiness, other meiotic abnormalities in these accessions are seen in the form of unoriented bivalents at metaphase-I, chromosomal bridges and laggards at anaphases and telophases (Figs. 15–19, Table 2) along with abnormal microsporogensis in the form of monads, diads and triads with or without micronuclei (Figs. 20–22). All these anomalies noted in these accessions seem to be leading to the formation of heterogenous sized fertile along with sterile 386 S. M. Jeelani et al. Cytologia 75(4) pollen gains and ultimately reduced pollen fertility (Figs. 23, 24). From all these studies, it is quite evident that irregular chromosomal variations appear to have contributed a lot in the speciation and evolution of the genus Impatiens.
Acknowledgements The authors are grateful to the University Grants Commission, New Delhi for providing financial assistance under the DRS SAP II and ASIST programmes. We are highly thankful to the Director BSI, Dehra Dun for their help in the identification of the plant species.
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