Chapter 3 Materials and methods

Transcription

1 Chapter 3

2 3. MATERIALS AND METHODS 3.1 M aterials The experimental plant species of the present investigation belong to the family Asclepiadaceae and are available in the North Eastern regions of India. They belong to three different genera viz., Calotropis, Asclepias and Hoya. The different experimental species of Calotropis, Asclepias and Hoya along with the places of their collection are as follows: Name of the Plant species Places of collection (i) Calotropis gigantea Br. Different places o f Goalpara, Abhayapuri, Bongaigaon o f lower Assam as well as from nearby areas of greater Guwahati. (ii) Calotropis procera Br. -do- (iii) Calotropis acia Hamilt. Goalpara and Bongaigaon area of lower Assam. (iv) Asclepias curassavica Linn. Sorbhog and Pathsala areas of Barpeta district, Lower Assam. (v) Hoya parasitica Wall. Gauhati University Campus and Basistha area of Kamrup district, Sibsagar and Golaghat o f Upper Assam. (vi) Hoya arnottiana Wight, -do- 3.2 Methods The collected plants were maintained in the Botanical garden, Department of Botany, Gauhati University, Assam. A set of plants from each species were also raised in earthen pot filled with well prepared garden soil to facilitate harvesting of root tips.

3 General taxonomic study Taxonomical investigations of the experimental plant species and their identification were carried out following Bentham and Hooker (1883), Hooker (1885) and Kanjilal and Bor (1939) Karyomorphological study Cytological investigations were made in the meristematic cells of the root tips in each plant species. Detail Karyomorphological studies include the following parameters; (i) Determination of somatic chromosome number (2n), (ii) Total length of the individual chromosome, (iii) Length of the long arm, (iv) Length of the short arm, (v) Position o f the centromere, ' (vi) Centromeric index, (vii) Relative length of the chromosome, (viii) Classification of chromosome. (ix) Volume o f chromosome. (x) Total form percentage (TF%). Details of procedure for measurement of each of the above parameters are described in the section Cytological and karyomorphological investigations. Collection of root tips To facilitate easy collection of root tips, seedlings of all the experimental plants were raised in earthen pots filled with well prepared garden soil. The root-tips were collected between 5 AM to 7.30 AM in the morning.

4 26 The collected root tips were washed and bleached thoroughly with running distilled water for 4 to 6 times to remove the latex. The root tips were then fixed in Camoy s solution (Glacial acetic acid 1 part + Absolute ethanol 3 parts) for 8 hours and finally stored in 70% ethanol). Squash preparation Hydrolysis and staining of chromosomes Root tips were first hydrolyzed by placing them in 0.1 N HC1 solution in a test tube for 15 minutes. During the hydrolysis process the root tips were gently warmed up. After hydrolysis, the root tips were placed in freshly prepared 2% acetocarmine (with iron) solution for 45 minutes to 1 hour for staining. During staining, the root tips were gently warmed up along with the staining solution and then allowed to cool. Root tips were then squashed in a drop of 45% glacial acetic acid in a clean grease free slide. Well spread metaphase stages were considered for the study. At least 10 such slides with well scattered metaphase plates were scored for karyomorphological study. Method for making temporary slide permanent Two different procedures were followed for making temporary squash preparations permanent. Procedure I: Permanent mounting procedure with separation of slide and covers lip (Anon. 1992) (i) At first separation of slide and coverslip have been done with proper marking on the slide. The setup was then inverted in a laverron box and

5 27 immersed in 45% acetic acid. The coverslip were separated from the slides and fall to the bottom of the box. Dyhydration of slides and coverslips were done by immersing in two successive washes in pure ethanol for 5 minutes each. The slides were taken in a borel tube and the coverslips in a petridish maintaining the same position as was in the Laverran box. Drying of slides and coverslips have been done separately at room temperature for at least 48 hours. After drying, a drop of euparol was placed on the upper surface of the slide at the centre and a fresh clean coverslip was turned over on to it restoring the initial marked position on the slide. Like wise the dried cover slip was also placed on a clean slide with a drop of euparol. The excess preservation was removed from the slide and the mount was allowed to dry at room temperature for at least a week. The setup was then cleaned with cotton saturated with alcohol. Procedure I I : (i) Paraffin seal in the temporary slide was removed and the slide was then inverted in a glacial acetic acid-butyl alcohol (1:1) mixture in a coupling jar till the cover glass is detached. (ii) Both the slide and the cover glass were then dehydrated by passing through two changes of absolute butyl alcohol keeping minutes in each. (iii) A drop of euperol was then put on the slide (at place where squash was made) and a fresh cover glass was placed over it.

6 28 (iv) In a clean grease free slides a drop of euperol was put and the detached cover glass (with squash and dehydrated) was placed properly. The permanent slides, thus prepared, become ready for cytological investigations. Length o f the individual chromosomes and their respective arms were measured with the help of ocular micrometer. Microphotographs of chromosomes were taken with the help of Olympus Camedia C5060 model microphotographic camera. Details of different criteria considered for karyomorphological analysis are described under the section Cytological and karyomorphological investigations Study of epidermal morphology Details of epidermal morphology of leaves were investigated in all the experimental plants species. For this purpose following four characters were considered. (i) (ii) (iii) (iv) Nature o f epidermal cells. Nature o f stomata. Stomatal frequency and index. Nature o f hairs and trichomes. The epidermal peels of both upper and lower surfaces of the leaves were made by mechanical stripping. These peels were stained with 2% aqueous saframin and mounted in 5% glycerin solution. Now the slide is ready for observation. The semi permanent slides thus prepared were scored under compound microscope (15x40x).

7 29 The nature of stomata and their subsidiary cells and epidermal cells have been studied. Number of stomata and epidermal cells were counted and stomatal frequency and stomatal index were calculated by using the following formulae. S Stomatal frequency (S.F.) = x 100 E c Stomatal index (S.I.) = x 100 E + S Where S = Average no. of stomata and F = Average no. of epidermal cells in the same unit area (Salisbury, 1927). The length and breadth of stomata were measured with ocular micrometer under high power (15x40x). Fifty readings for each character were taken. The mean values were calculated, statistically analysed and the results were tabulated. Microphotographs of both upper and lower epidermal peels were taken with the help of Olympus Camedia C5060 model microphotographic camera. In describing the epidermal features, terminology for epidermal cells and stomata were classified and described as suggested by Rajagopal and Ramayya (1981). The stomatal index and frequency were calculated by the formulae given by Gupta (1961) and Salisbury (1927) respectively Study of morphology of pollinaria and pollen grains For the study of morphology of pollinaria and pollen grains, fresh flower buds were collected from healthy plants of all the six experimental species. Collected flower buds were preserved in 70% ethanol. The pollinaria were isolated under a dissecting microscope (lox) and outer morphological features were studied properly. For detailed morphological study the following criteria were considered:

8 ^ 30 (i) (ii) (iii) (iv) (v) Shape, colour and attachment of the pollinia, Length and breadth of the pollinia, Length and breadth of the translator, Length and breadth of the corpusculum, Length and breadth of the caudicle. The different parts of pollinaria were measured under a compound microscope (4x1 Ox) by using an ocular micrometer. For the study of morphological features in pollen grains the pollinia were dissected and by following the procedure of Erdtman (1952, 1960) and Nair (1961) the permanent preparation of pollen grains were prepared. Detail procedure for making pollen slide Isolated mature pollinaria collected from fresh flowers were dipped in 70% ethanol for about an hour. After crushing the pollinia the suspension was poured through a brass metal net (100p meshes) into centrifugal tubes and centrifused for about 3 minutes and the alcohol was decanded. The sediment was then washed with 45% glacial acetic acid, crushed again in 45% glacial acetic acid and poured through net to centrifugal tubes. The centrifugal tubes containing the polliniferous material were again centrifugated and the acid was decanted off. Then the acetolysis mixture was prepared with 9c.c. of acetic anhydride and lc.c. of concentrated sulphuric acid. 5c.c. of thus freshly prepared acetolysis mixture was then added to the sediment gently. The tubes containing pollen grains were then transferred to water bath and warmed up to boiling point. The tubes were kept for another 10 minutes in the water bath itself to slowly cool down. The centrifugal tubes were then placed in ice cold

9 31 water. After centrifusing again for a minute the supernatant was decanted and the sediment was washed again with 45% glacial acetic acid and centrifuged. The glacial acetic acid was finally decanted and the residue was washed with distilled water, the centrifused and supernatant was discarded. Microscopic slides of pollen grains were prepared in gelatin-jell. Microphotographs were prepared using Olympus camera. The size of the pollen grains were measured with the help of ocular and stage micrometer. Mean value o f ten readings for each type was calculated for respective length and breadth. Polar view P/E [= ratio was also calculated (P=Polar view, E=Equatorial Equatorial view view). Length and breadth of pollinaria, translator, corpusculum and caudicle were measured and the data were used for comparative studies amongst the experimental species. 3JL5 Study of seeclmorphology Viable matureand dry seeds of the experimental plant species were freshly collected from the naturally dehiscing follicles during the year The seeds of all the species were observed under a dissection microscope (lox) while their hairs and testa were studied under a compound microscope (loxlox). Weight of one hundred seeds have been obtained from a microbalance. The colour of the seeds, length and breadth of the seeds, total number of seeds in a follicle were also recorded. The hairs or coma of all the species have been studied and the length o f the coma were also measured. Ten reading for each characters were taken and the mean values were used for statistical analysis.