Introduction

Tabernaemontana, a member of Apocynaceae, is distributed in tropical countries including Brazil, Egypt, India, Sri Lanka, Vietnum, Malayasia and Thailand.

Tabernaemontana dicotoma is evergreen dichotomously branched large tree (upto 8 m) with oblong leaves, and star shaped flowers borne in cyme. The petals are in single whorl and it flowers from March to June. Fruit is a pair of follicles and curved with pointed ends [3, 26]. T. coronaria is evergreen shrub with elliptic and shining green leaves. The flowers are arranged in cyme. T. coronaria var. variegata, is a medium size plant with variegated leaves and T. coronaria var. Dwarf, on the other hand, is a small and compact plant. T. coronaria var. Flore-pleno is a large plant with comparatively larger leaves, and double flowers [3].

The leaves and bark of T. dicotoma are purgative. The milky sap is also said to possess cathartic properties. A crystalline alkaloid, non-phenolic in nature and soluble in ether, has been isolated from roots [26]. Tabernaemontana coronaria is a medicinally important plant with anti-ulcer, anti-bacterial and anti-inflammatory properties [1, 7, 14, 23]. This plant is a rich source of different alkaloids including indole alkaloids. Phytochemical studies reveal presence of many indole alkaloids, non- alkaloid constituents such as enzymes, flavonoids, hydrocarbons, phenolic acids, phenyl propanoids, steroids and terpenoids [15]. Tabernaemontana is used in Ayurveda, Chinese and Thai traditional medicine for the treatment of various diseases [2, 24].

The chromosome number in different species and varieties of Tabernaemontana has been found to be 2n = 22 except in T. coronaria var. Flore-pleno (with 2n = 3X = 33 chromosomes) [5, 17, 18, 19], having basic chromosome number x = 11. The estimation of nuclear DNA content in situ is also considered as a useful tool to study evolutionary patterns and affinity in higher plants [20]. It has been observed that the amount of nuclear DNA varies even between closely related species within the same genus from three to six fold [4, 21]. There are also records where different species of the same genus do not necessarily show a major difference in DNA content [6, 16].

The present investigation was undertaken to analyze karyotypes and in situ DNA contents of T. dichotoma, T. coronaria var. variegata, T. coronaria var. Dwarf, T. coronaria and T. coronaria var. Flore-pleno to study genetic diversity among species and varieties of Tabernaemontana.

Materials and methods

Three varieties and its populations of Tabernaemontana coronaria and T. dichotoma were selected for morphological and cytological investigations. The list of species and their collection details are as follows: (i) Tabernaemontana dichotoma Roxb. ex Wall. [Population I- AHSI, Kolkata, (22.566 °N and 88.3667 °E)], (ii) T. coronaria var. variegata [Population I- AHSI, Kolkata, and population II- Howrah (22.5900°N and 88.3100°E], (iii) T. coronaria var. Dwarf [Population I- AHSI, Kolkata, and population II- Howrah], (iv) T. coronaria (Jacq.) Willd. [Population I- AHSI, Kolkata, and population II- Howrah, population III- Hooghly, (22.8956°N and 88.4025°E) and population IV- Midnapore, (22.4240°N and 87.3190°E)], (v) T. coronaria var. Flore-pleno [Population I- Howrah].

Plants were grown in experimental garden of University of Calcutta. The plants were taxonomically identified by the experts of Central National Herbarium, Shibpur, Howrah.

Morphological study

The different morphological characters selected were inter-node length, petiole length, leaf area and leaf index. Twenty five readings were taken at random for each plant and mean values as well as standard errors were calculated.

Study of somatic chromosome

Somatic chromosomes were studied from root tip Cells. maximum meristematic activity was found to be between 10:40 AM to 12:30 PM. After collection, roots were pretreated in a mixture of super-saturated aqueous solutions of p- dichlorobenzene (PDB) and 2.0 mM 8- hydroxyquinoline (1:1). Pretreatment was carried out at 18 °C for 5 h, after an initial shock treatment at 0 °C for 5 min [9] and then fixed in chilled Carnoy’s fixative at 18 °C for overnight. The root tips were hydrolyzed in 1 N HCl for 12 min at 60 °C. Finally root tips were stained in 2 % propionic-orcein at room temperature (22 °C − 23 °C) for 3 h and squashed in 45 % propionic acid.

Chromosomes were classified into different types based on their ‘i’- values [10]. Chromosome volume was calculated using the formula: chromosome volume (v) = πr2h, where r = radius of the chromosome = breadth/2 and h = whole length of the chromosome [11, 13].

In situ nuclear DNA content estimation

For microspectrophotometric in situ nuclear DNA estimation, pretreated and fixed root tips were hydrolyzed in 1 N HCl for 10 min at 60 °C, followed by Feulgen staining (Schiff’s reagent) for 1 h. at 18 °C in dark. The root tips were squashed in 45 % acetic acid. Cytophotometric analysis was carried out by critically examining about 25 late prophase to metaphase stages from different root tips of each plant with a microspectrophotometer at a wavelength of 550 nm [22]. The amount of nuclear DNA was measured in arbitrary units of relative absorbance on the basis of optical density. The relative arbitrary units of absorbance were converted to absolute units (picogram) by considering the 4C nuclear DNA amount of Allium cepa var. rosette (67.1 pg) as the standard [25].

Results and discussion

The species and varieties of Taberbaemontana differed with respect to leaf size, inter node length and petiole length (Table 1; Fig. 1). Length of petiole varied from 1.5 ± 0.5 mm in T coronaria var. Dwarf to 15.0 ± 1.7 mm in T dichotoma. Leaf index value ranged between 2.66 ± 0.09 in T. coronaria var. Flore-pleno and 4.99 ± 0.71 in T dichotoma. However, the leaf indices did not differ much in T. coronaria, T. coronaria var. variegata and T coronaria var. Dwarf, though remarkable variation was noted in the petiole length in T coronaria var. Dwarf. Length of petiole and leaf area are distinct from each other and may be considered as identifying characters of species and varieties of Tabernaemontana. Length of inter-node did not differ much among these species and varieties except in T coronaria var. Dwarf. The diploid plants differed markedly in morphological characters. The triploid variety of T coronaria T. coronaria var. Flore-pleno showed higher values in both vegetative and floral characters than diploids (Fig. 1) indicating the fact that increase in leaf and flower size is associated with polyploidy. Petiole length and inter-node length did not correlate with polyploidy. However, the constancy in values of morphological characters in species and varieties of Tabernaemontana indicates genetic control [19].

Table 1 A comparative representation of morphological features in species and varieties of Tabernaemontana
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Fig. 1
figure 1

Photograph of plants: a. T. dichotoma Population I, b-c. T. coronaria var. variegata Population I-II, d-e. T. coronaria var. Dwarf Population I-II, f-i T. coronaria Population I-IV, j. T. coronaria var. Flore-pleno Population I

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The somatic chromosome number was found to be 2n = 22 in T. dichotoma, T. coronaria T. coronaria var. variegate and T. coronaria var. Dwarf whereas in T. coronaria var. Flore-pleno, somatic chromosome number was 2n = 3× = 33 (Table 3; Fig. 2) as also reported earlier [19]. Chromosomes are mostly medium in size ranging between 1.45 μm to 4.37 μm in length, but in Dwarf variety it ranges from 1.45 μm to only 2.12 μm (Table 3). Chromosomes of all the species and varieties of Tabernaemontana studied here revealed a gross morphological similarity. The seven different chromosome types were noted to be common among these species and were classified on the basis of number and position of the constrictions (Table 2). In general four types of nucleolar and three types of centromeric chromosomes were found (Table 2). The chromosome number and types varied among the species and varieties of Tabernaemontana.

Fig. 2
figure 2

Photograph of somatic metaphase plates and drawing of the same plates (bar = 5 μm): a-b T. dichotoma Population I, c-f T. coronaria var. variegata Population I-II, g-j T. coronaria var. Dwarf Population I-II, k-r T. coronaria Population I-IV, s-t T. coronaria var. Flore-pleno Population I

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Table 2 Different types of chromosome present in different species and varieties of Tabernaemontana
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The total chromosome length (TCL) varied from 35.95 μm in T. coronaria var. Dwarf to 102.93 μm in T. coronaria var. Flore-pleno. Among the diploids, the highest total chromosome length was obtained in T. dichotoma and least was found in the variety Dwarf. Minute differences were observed in population level.

The total chromosome volume (TCV) varied from 33.64 μm3 in T. coronaria var. Dwarf to 109.76 μm3 in T. coronaria var. Flore-pleno. The total chromosome volume was also least in Dwarf variety and highest in T. dichotoma (Table 3). The total chromosome volume differed remarkably in T. dichotoma, T. coronaria T. coronaria var. variegata, T. coronaria var. Dwarf with similar chromosome number. This indicated the possible role of differential condensation of the chromatin and association of both histone and non-histone proteins [8, 9, 21]. A conspicuous variation was observed in population level too.

Table 3 A comparative representation of different chromosomal parameters in species and varieties of Tabernaemontana
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Karyotype formula was expressed numerically based on chromosome type and number of chromosomes in each type (Table 3; Fig. 3). The species and varieties showed a graded karyotype with are medium to short chromosomes. The number of chromosomes with nucleolar constrictions varied from 6 to 12 in these species and varieties of Tabernaemontana. Duplication of chromosome or translocation between the chromosomes with or without secondary constrictions might be responsible for the increase or decrease in number of secondary chromosomes [12, 13]. Each species and varieties has specific karyotype, total chromosome length and total chromosome volume indicating their distinctiveness. Karyotype formula did not differ at population level. The results have clearly revealed that minute structural alterations in chromosomes played important role in the evolution of these species and varieties of Tabernaemontana [13, 19]. These characters which are under genetic control can be utilized as parameters for classification and identification of species and varieties [13].

Fig. 3
figure 3

Karyogram (bar = 5 μm), a.T. dichotoma Population I, b-c. T. coronaria var. variegata Population I-II, d-e. T. coronaria var. Dwarf Population I-II, f-i. T. coronaria Population I-IV, J. T. coronaria var. Flore-pleno Population I

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The mean OD value at late prophase to metaphase of Allium cepa var. rosette was found to be 35.6 which corresponds to 67.1 pg of 4C nuclear DNA. The 4C nuclear DNA contents in the present study varied from 21.60 pg to 33.40 pg (Table 4). Nuclear DNA content was correlated positively with total chromosome length and total chromosome volume. Intra-specific and intra varietal constancy in the amount of nuclear DNA was also recorded. Among the diploids, the highest 4C nuclear DNA value was obtained in T dichotoma (25.30 ± 1.11 pg) and least was found in T coronaria var. Dwarf population II (21.60 ± 2.11 pg) (Table 4). The other varieties and populations of T coronaria showed intermediate values. The consistency in the amount of 4C DNA at late prophase and metaphase stages of species and varieties studied has been recorded. Therefore, the consistency and difference in the amount of nuclear DNA in species and varieties of Tabernaemontana can be utilized as identifying parameters. The 4C nuclear DNA value did not differ much at intra specific and intra varietal level, suggesting an adaptive value [8].

Table 4 Chromosome number and 4Cnuclear DNA content in species and varieties of Tabernaemontana
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