ABSTRACT- Karyomorphological studies in Bauhinia acuminata Linn. and Bauhinia variegata Linn. belonging to family Caesalpinaceae using leaf tip squash technique and induction of polyploidy in Bauhinia acuminata Linn. was carried out. It was observed that in the Karyomorphological studies both the species have the same number of chromosomes i.e., 2n=28 with chromosome length showing symmetric karyotype, with largely submetacentric and metacentric chromosomes. On induction of polyploidy in Bauhinia acuminata Linn. the result showed that the maximum induction of polyploidy was obtained in the case of seeds treated with 0.10% concentration of colchicine for consecutive 3 days. Karyotype analysis of the induced polyploidy revealed that the chromosome number was obtained 4n=56, tetraploid. The significance of polyploidy showed increase in plant height, leaf size and stomata size of the plant under investigation
Key-words- Karyomorphology, Induction of Polyploidy, Colchicine, Tetraploid

INTRODUCTION
Genus Bauhinia has great medicinal value and is the largest genera of the tribe Bauhinieae in the family Caesalpinaceae under De Wit's revised system of classification, 1956 which represents more than 300 species. Bauhinia genus consisting of trees, climbers and shrubs are distributed in India [1] of which 12 spe-cies are being reported from North Eastern regions of India [2]. The simple bilobed leaf is an outstanding character of this genus principally responsible for its separate tribal status. This character is not only con-spicuous but is universally present in all the species with slight variations in the extent of lobation. Taxo-nomically it is regarded as a uniform genus by all tax-onomists regarding it as a natural assemblage.
In this study, two closely related species of Bauhinia of the same family viz., B. acuminata Linn. and B. variegata Linn. were selected. Both the plants are ethnomedicinally important plants of Assam [2].
B. acuminata Linn. commonly called as Mati-katota in Assamese is not so common species and found in Rani Reserve- Kamrup, North Cachar Hills- North Range, Bhumeswar Hill-Goalpara and often cultivated in gar-dens of various parts of Assam, India. Commonly known as Dwarf White Orchid Tree is widely cultivated for its ornamental value. These species occur widely in deciduous forest and scrub. Leaves are bilobed, shaped like an ox or cow hoof; long and broad, with the apical cleft. Flowers are fragrant, with five white petals, ten yellow tipped stamens and a green stigma. Fruit is a pod 7.5 to 15 cm long and 1.5 to 1.8 cm broad. Perfect little tree for places where we don't want anything wild to take over [3-5].
B. variegata Linn. commonly known as (orchid tree, camel's foot, mountain ebony, Napoleon's hat, paper mulberry, poor man's orchid, bauhinia); locally called Bogakatra, Kurol, Kotora in Assam. Throughout the province ascending to 2500ft in the Khasi Hills, more common in hilly regions. B. variegata Linn. is small to the medium-sized deciduous erect tree. Leaves as broad as long or sometimes broader usually deeply cordate. Flower clusters are unbranched at ends of twigs, buds not angled, fertile stamens 5. They are capable of growing on a wide range of soils from gravelly, shallow, rocky soil on hill slopes to sandy loam and loamy soil in the valley.
Leaves make good fodder and eaten by sheep, goats, and cattle. The main uses of Bauhinia plant are as fuel calorific value is 4800 kcal/kg [6]. The mature seeds and young pods of Bauhinia are eaten, cooked and pickled in the native countries. The Bauhinia leaves extract is being used for medicinal purposes including anti-inflammatory, an-tifungal, antipyretic, analgesic, antispasmodic, antitumor and antimicrobial activities [7]. The stems, roots, and leaves are also used for the treatment of several diseases especially in pain, diabetes, infections, ulcer, jaundice, leprosy and also utilized as folk medicines [8]. Phytochemical study of bark extract revealed the presence of flavonoids which have anticarcinogenic activity. The plant extract of Bauhinia variegata Linn. due to the presence of â- sitosterol exhibited a significant hypolipidemic effect, reduced the obesity as well as decreased the levels of cholesterol, triglyceride, VLDL holesterol (lipid profile) [9]. Lectins (glycol proteins) from Bauhinia seeds have been reported to possess antitumor activity [10]. The Bauhinia seeds are known to be a good source of protein, vitamin A, and minerals. It also contains a significant amount of oil as compared to soya bean and cotton seeds nutritionally viable for human health [11].
Bauhinia is a plant of great economic importance. Since some anticarcinogenic properties have been found especially in B. variegata Linn. thus this species serves as a raw material for lead molecules in anti-carcinogenic drug development. As such it needs conservation and detailed chromosome study would aid in creating germplasm.

MATERIALS AND METHODS
This study was conducted in the Department of Botany, Handique Girls’ College, Guwahati (Assam), India in the duration of six months from January- June 2016. Seeds of Bauhinia acuminata Linn. were collected from the Handique Girls’ College Campus, Guwahati, Assam and the seeds of Bauhinia variegata Linn. were collected from Amsoi Hills, Nagaon, Assam and were raised under department botanical garden.

Method of Cytological investigation- For detail karyotypic studies, karyotypes were prepared from the somatic chromosomes. For cytological studies, leaf tip squash technique was done. Very young leaves were collected from the B. acuminata Linn. and B. variegata Linn. between 7:00 a.m. to 7:30 a.m; washed in double distilled water and pre-treated with saturated solution of paradichlorobenzene at a suitable temperature for 3 hours at 4°C ± 2°C. Pretreated young leaves were fixed in a suitable fixative such as Carnoy’s Fluid-II (1:3:6; Glacial acetic acid: Chloroform: Ethanol) for 24 hours to 28 hours at room temperature. After fixation, the leaves were thoroughly washed with 70% ethanol and finally, they were stored and used for cytological work. For the preparation of slides of leaves were firstly stained with acetic orcein: nHCL (9:1) mixture and warmed over the flame for 10-15 mins and kept for 2-3 hours at room temperature. Single leaf tip was taken in a drop of 45% acetic acid on a slide. Only the dividing tip region was taken discarding the other tissue. Coverslip was placed over the tip and squashed by applying uniform pressure with the thumb through a piece of blotting paper, a gentle tapping is done with matchstick after passing it lightly over the flame and finally sealed with paraffin for further studies [12]. The temporary slides thus prepared were observed under the compound microscope at a magnification of 1000x using oil immersion (10×100x, oil immersion). This procedure was standardized through trial and error method.
Well, scattered metaphase plates were selected for karyomorphological analysis of the chromosomes. Perfectly stained chromosomes were photographed using trinocular microscope N400-M, CMOS camera with image analysis system. The drawings of the chromosomes were made with the help of camera lu-cida apparatus. Idiograms were then constructed on tracing paper.
Following parameters of the chromosomes were considered:

(i) Length of the long arm (ii) Length of the short arm (iii) Total length of the chromosome (iv) Volume of the chromosome (v) Relative length and (vi) Centromeric position.
On the basis of the length, the chromosomes were categorized under the following type.
Type A = 3.00µm and above, Type B= 2.50µm to 2.90µm, Type C= 2.00µm to 2.49µm, Type D= 1.00µm to 1.90µm, Type E= 0.01µm to 0.99µm.
The volume of an individual chromosome was calcu-lated as Chromosome Volume (V) = pr²h.
Where, r= radius of the chromosome h= length of the whole chromosome. The total chromosome volume was then expressed by adding the volumes of all the chromosomes of the complement [17].

Induction of Polyploidy in Bauhinia acuminata Linn.- Induction of polyploidy was undertaken in Bauhinia acuminata Linn. using colchicine as the polyploidizing agent. For these purpose, four different concentration as 0.05%, 0.1%, 0.25%, 0.50% were used. For preparing each of these concentrations 0.05gm, 0.1gm, 0.25 and 0.50gm colchicines powder were dissolved in 100cc of double distilled water respectively.
For induction of polyploidy in Bauhinia acuminata Linn. seeds were treated with colchicine solutions. For this purpose, mature seeds were collected and properly dried prior to colchicines treatment. These seeds were soaked in dou-ble distilled water for 6 hours. The pre-soaked seeds were used in each treatment. Treatments were started from 8 a.m. in the morning to colchicine with the chromosome divisional cycle. The seeds were treated with 0.05%, 0.1%, 0.25% and 0.50% colchicine solution for 3 consecutive days with 12 hours interval. After treatment, the seeds were washed thoroughly with double distilled water to eliminate the residual effects of the chemical if any. Treated seeds and stem cuttings were then planted in earthen pots filled with a mixture of well-prepared soil, farmyard manures and sand in the ratio of 3:1:1. Root and shoot initiation started after 8 and 15 days of treatment respectively. Plants does risen were tested morphologically as well as cytologically for polyploidy. Cytological assays comprise of chromosome count in the root meristematic cell. Root tip squash was prepared and Karyomorphological analysis was done. Morphological assay for induced polyploidy plant height, leaves size, petiole length, internode length and size of stomata its frequency. These parameters were studied in the treated plants, when they become 45 days and elder and were compared with those of normal diploid plants.
For the study of the stomata, epidermal peels of the lower epidermis of the leaves were taken out by mechanical stripping stained with safranin and finally mounted in 4% glycine by taking in a clean slide. The slide thus prepared was observed under the compound microscope (10× 40x). The number of stomata and epidermal cells was counted. The stomatal frequency and stomatal index were calculated using the following formula.

Stomatal Frequency (S.F) = S / E X 100

Stomatal Index (S.I) = S / E+S X 100

Here, S=Number of stomata , E= Number of the epidermal cell
The length and breadth of stomata were measured with an ocular micrometer (10×40x magnification). Lower epidermal peels were microphotograph using trinocular microscope-N400-M, CMOS camera 5M with image analysis system.

RESULTS
Cytological and karyotypic investigations in B. acuminata Linn. and B. variegata Linn. were showed that both the species consist of 2n=28 chromosomes (Table 1). On the basis of the length the chromosomes it was observed that B. acuminata Linn. six chromosomes belong to type A and twenty-two chromosomes belong to type B and the chromosome formula can be written as: A₆ + B₂₂+C₀+D₀+E₀=2n=28. Depending on the position of their centromere, the karyotypic formula can be written as M₆+Sm₂₂=28. However, in B. variegata Linn., it was observed that twenty-four chromosomes belong to type D and four chromosomes to type E. So the chromosome formula can be written as: A₀+B₀+C₀+D₂₄+E₄=2n=28 and karyotypic formula can be written as M₄+Sm₂₄=28.
Induction of polyploidy in Bauhinia acuminata Linn. revealed, all the concentrations of the colchicines were not equally effective in inducing polyploidy. Colchicine solution having concentration 0.10% was found to be most effective. Karyomorphic analysis of the induced polyploidy revealed the chromosome number to be 56 while in the case of the diploid the somatic chromosome number was found to be 28 (Table 1). Out of these 56 chromosomes, a notable difference between the longest and the shortest chromosome was also seen. A general increase in the chromosome size was noticed when compared with those in the diploid. The length of the 56 chromosomes was measured and found to vary from 0.70µm to 2.4µm whereas, those in the case of the diploid varied from 1.00µm to 1.5µm. It has been observed in induced polyploidy that since a lot of chromosomes are present, depending upon their length the chromosomes four chromosomes were categorized under type C, a total of fifty chromosomes were observed under type D out of which forty-six chromosomes were categorized under subtype d/ and six chromosomes were under subtype d// and two chromosomes are categorized under type E. Based on this the chromosome formula and karyotypic formula can be written as: C₄+D₅₀(d_/42+d//₈)+E₂ = 4n= 56 and M4 + Sm₅₀ + St₂= 4n = 56 respectively.
The microphotograph, camera lucida diagram, karyo-type and idiogram of the chromosomes are represented in Fig. 1 (A), (B), (C), and (D) respectively.





Fig. 1: (A) Microphotograph of somatic chromosomes at metaphase stage (15× 100x oil immersion);
(B) Camera lucida diagram of somatic chromosomes at metaphase stage (10 × 100x oil immersion);
(C) Karyotype;
(D) Idiogram (a) B. acuminata (b) B.variegata (c) Induced polyploidy in B. acuminata

Table 1: Summary of the karyotype in the normal diploid (2n) and induced tetraploid (4n) of B. acuminata Linn.

Taxa	Chromo-some No. (2n)	Range of chromosome					Types of chromosome	Karyotype formula
		Length	Relative length	Radius	Volume	Arm ratio
B. acuminata Linn.	28	1.00-1.50	50.00-60.00	0.40	0.50-0.90	1.00-1.50	A6+B22	M6+Sm22 2n=28
B. acuminata Induced polyploidy	56	0.70-2.4	51.00-80.00	0.50	1.25-1.50	1.00-4.00	C4+D50(d/42+d//8)+E2	M4 + Sm50 + St2 =4n=56

DISCUSSION
A detailed survey was undertaken to ascertain the uses of these plants for treatment of various by the ethnic people of North Eastern Region of India. Both the plants are ethno-medicinally important plants of Assam [2]. In the present investigation, it has been found that both the species are growing in different parts of the North Eastern regions of India and are variously used by the ethnic people of the region. The flower is used to cure diarrhea and leaves and flowers are eaten in North-Eastern regions of the country [8]. Naga uses the plant as an antidote to certain toxins and poisons [13]. Bauhinia acuminata Linn. had high SPF value with antioxidant, antibacterial property and can be used as an efficient agent for UV radiation hazards [14]. Contains a significant amount of oil as compared to soya bean and cotton seeds nutritionally viable for human health [11]. Lectins (glycol proteins) from Bauhinia seeds have been reported to possess ntitumor activity [10]. The presence of flavonoids (anticarcinogenic activity) [15]. Anti-inflammatory, antifungal, antipyretic, analgesic, antispasmodic, anti-tumor and antimicrobial activities [7]. Good source of protein, vitamin A and minerals [11]. Keeping these important medicinal aspects in mind, commercial cultivation of such remarkable and useful plants as a raw material for the development of new drug formulations in the treatment of life-saving ailments in the near future may not be ruled out. Such a significant plant deserves greater attention, conservation, and protection.

Karyomorphology- The importance of karyotype analysis in distinguishing plants species is well known. Karyomorphology and chromosome number of a variety or species are useful in its identification as also in establishing the evolutionary relationships among related species [16]. Karyotype suggests primitive and advanced features. In the present investigation, a comparison has been made between the karyotypes of two species of Bauhinia Linn. through leaf tip squash method may, therefore, be considered as the first attempt through this technique to study the detailed cytological aspects.
In both B. acuminata and B. variegata the chromosome number is constant with 2n=28 and chromosome morphology is grossly identical in all cells. The cyto-logical study reveals that the karyotypes are symmetric which signifies primitive characters. A gross resemblance in the nature of the karyotype in rather short chromosomes with gradation in size but with no abrupt size difference in the complements. A detailed graphical representation summarising the complete karyotypic analysis, shown in Fig. 2.



Fig. 2: Graphical representation of the comparative analysis in the normal (2n) diploid and
induced (4n) tetraploid in B. acuminata Linn.
Polyploidy- Karyotype studies on induced tetraploid revealed the somatic chromosome number to be 4n=56 whereas, in the normal diploid plant it was found to be 2n=28. A general decrease of the chromosome size was noticed when compared with those in the diploid. The doubling of the chromosomes produced significant increases in the plant height, leaf size and stomata size (Fig. 3). The findings of the present studies reveal that the ploidy level greatly influenced the size of the stomata. The significant reduction in the stomatal frequency/ sq.mm observed in these polyploidy plants is indicative of the consequence of stomatal expansion caused by an increase in their sizes (Table 2).



Fig. 3: Morphology of plant, leaves and stomata in normal diploid (2n) and induced tetraploid (4n) in
Bauhinia acuminata Linn.
(a) Photograph showing plant height in normal (2n) Induced tetraploid (4n)
(b) Photograph showing leaf size in normal (2n) and induced tetraploid (4n) seedling after 8-10 days of
germination
(c) Photograph showing stomata in normal diploid (2n)
(d) Photograph showing stomata in induced tetraploid (4n)

Table 2: Details of stomatal characters in normal diploid (2n) and induced tetraploid (4n) of Bauhinia acuminata Linn.

Ploidy level	Stomatal index	Frequency of stomata/sq.mm	Length of stomata (L) (µm)	Breadth of stomata (B) (µm)	Area of stomata (L×B) (sq.µm)
Diploid	24.44	19.64	10.30±0.25	7.85 ± 0.04	10.30 × 7.85
Polyploid	15.55	13.46	22.50±0.23	16.50 ± 0.20	22.50 × 16.50

CONCLUSIONS
This study represents a detailed karyomorphological study undertaken in B. acuminata Linn. and B. variegata Linn. for their genetic improvement. Concerning their importance and utility in the production of flavonoids, lectins, proteins, vitamin A, minerals and a significant amount of oil as compared to soya bean and cotton seeds, ex-situ conservation measures should be initiated for protection, preservation, and regeneration of such remarkable medicinal plants. Further investigations into the chemical constituents of these plants may definitely lead to the development of new drugs. Induction of polyploidy increases induction of polyploidy increases the number of chromosomes and usually brings about an increase in the size of the affected cells and various degrees of changes in their functions. These polyploids can be useful for breeding programs and production of higher yields of secondary metabolites like flavonoids for economic, medicinal purpose and for driving evolutionary processes.

REFERENCES

1. C. Valdir. Chemical composition and biological potential of plants from the genus Bauhinia, Phytother Res, 2009; 23:1347-54.
2. U.N Kanjilal, P.C Kanjilal, and A. Das. Flora of Assam. 1938; 2:139-40.
3. Pacific Island Ecosystems at Risk (PIER): Bauhinia acumi-nata. Available from: http://www.hear.org/Pier/species/bauhinia_acuminata.htm.
4. Flowers of India: White Orchid Tree. Available from: http://www.flowersofindia.net/catalog/slides/Dwarf%20White%20Orchid%20Tree.html.
5. Huxley, A., ed. (1992). New RHS Dictionary of Gardening. Macmillan. Available from: https://www.coursehero.com/file/p1d2lf3/Huxley-A-1992-ed-New-RHS-Dictionary-of-Gardening-Macmillan-ISBN-0-333-47494-5/.
6. Shilpa Gautam. Bauhinia variegata Linn: All Purpose Utility and Medicinal Tree. Forestry Bulletin, 2012; 12(2):61-64.
7. Y.R Kumar and G.P. Rajani. Analgesic and Anti-Ulcer Activities of Ethanol and Aqueous Extracts of Root of Bauhinia Variegata Linn. Int. J. Pharmacol, 2011; pp:616-622.
8. S. Pendy and R. C. Agraval, Glob. J. Pharmacol., 2009; pp:158-162.
9. G. Balamurugan and P. Muaralidharan, Bangladesh J. Phar-macol., 2010; pp: 8-12.
10. A.Jose, C.S Silva Daniela, P.A Damico, M.A Baldasso, The Protein J.2007;193-201
11. Sarfraz Arain, et. al., Physico-chemical Characteristics of Oil and Seed Residues of Bauhinia variegata and Bauhinia lin-naei .2012; pp:16-21.
12. Nabis Bandana. Karyomorphological studies and 4C Nuc-lear DNA estimation in two species of Plumbago Linn. Proceedings of Assam Science Society, 2014; 15: 209-216.
13. Jain and De, Sharma, P.K., S.K Dhyani, and V. Shanker. Some useful and medicinal plants of the district Dehradun and siwalik. J. Sci. Res. Pl. Med. 1979; pp:17-43.
14. Jayanthy V, Shafna A, Use of flowers as anti-melanocyte agent against UV radiation effects. American J of Bio-pharm, Biochem, and life sci., 2012; A68.
15. B.Rajkapoor, B. Jayakar and N. Murugesh, J. Ethnopharmacol., 2003; pp:107-109.
16. Lavania UC and Srivastava S., Quantitative declination of karyotype variation in Papaver as a measure of phyloge-netic differentiation and origin. Curr. Sci. 1999; pp:429-435.
17. Levan A, Fredga K and Sandberg AA. Nomenclature for centromeric position on chromosomes. Heriditas 1964; 52(2):201-220.

How to cite this article: Basumatari M, Das BN: Karyomorphological Studies in Two Species of Bauhinia Linn. and Induction of Polyploidy in Bauhinia acuminata Linn. Int. J. Life. Sci. Scienti. Res., 2017; 3(4):1223-1229. DOI:10.21276/ijlssr.2017.3.4.20 Source of Financial Support: Nil, Conflict of interest: Nil