Zingiberaceae including Z. officinale. Sato (1960) carried out Karyotyping studies of 24 species belonging to 13 genera of the family Zingiberaceae and concluded that the basic number of the genus Zingiber is n = 11, and of Z. mioga with 2n = 55 is a pentaploid.
Ramachandran (1969) studied the cytology of five species of Zingiber (Z.
macrostachyum, Z. roseum, Z. weihtianum, Z. zerumbet and Z. officinale) and found a diploid number of 2n = 22 in all species. He found evidence of hybridity involoving interchanges and inversions in ginger. Mohanty (1970) studied the cytology of Zingiberales and reported 2n = 22 for Z. spectabile and Z. cylindricum and concluded that the genus Zingiber is closer to Hedychium. Das et al. (1999) investigated various cultivars of Zingiber and found that all of them possess a somatic chromosome number of 2n = 22. An asymmetrical karyotype of “1B” was found in all the cultivars except in cultivars of Bangkok and Jorhat, which have a karyotype asymmetry of “1A”. The karyotype of various cultivars exhibited only minor differences. However, in this study, ploidy level of Z. moran has been investigated for the first time.
packet. In addition to the above data, the field data ecology, associated smell and the colour of the rhizome, leaves, flowers, fruits and aerial colour and other details of the floral parts were noted.
1.3.2. Identification and maintenance of germplasm
The collected plants were then maintained as both live specimen and as herbariums in the departmental green house of Indian Institute of Technology Guwahati (IITG), Assam.
Herbaria are essential for the study of plant taxonomy, geographic distributions, and the stabilizing of nomenclature. The specimens in a herbarium are often used as reference material in describing plant taxa; some specimens may be types. To preserve their form and color, collected plants were spread flat on sheets of newsprint and dried in between absorbent paper. The specimens are then mounted on sheets of stiff white paper, labeled with all essential data such as date and place found, description of the plant, altitude, and special habitat conditions. The sheet was then placed in a protective case. As a precaution against insect attack, the pressed plant is frozen or poisoned and the case disinfected.
A botanical specimen consists of the whole plant, complete with roots, stem, leaves, flowers and if possible, with fruits. Since Zingiberaceae species were large herbs, a portion of a twig with leaves and flowers were collected as a representative specimen for herbarium. The size of the specimen used for preservation was set according to the size of the herbarium sheet (17x11 inches) used for mounting. After collection, the specimen was tied with a tag number. Most of the plants wilted very rapidly after being cut or dug out of the ground. To avoid this, the dug out plants and cut twigs were kept in polythene bags along with some soil retained with the rhizome. This could keep the plants fresh for more than 6 hrs. The flowers of C. amada were too large to be pressed with the leaves and were kept separately in small polythene bags. Two specimens from each were collected for every species. Soon after collection, pressing of the specimens was carried out with a ventilated drying press. While pressing, care was taken so that the leaves are upside down to show the ventral side. Rhizomes were difficult to press satisfactorily and hence were dried separately with tags. The specimens were examined in every 24 hrs. The newspapers were replaced with new ones in every week. Next, the plant twig was mounted to a herbarium sheet and was labeled. A label consisted of
collection number, Latin name, family name, vernacular name, habit, habitat, locality, date of collection and collector's name. Then the herbarium sheets were stored under a cover, called species cover. Collected specimens were identified by studying morphological traits following taxonomic rules and with the help of herbarium of Gauhati University.
1.3.3. Ploidy check by chromosome study 1.3.3.1. Collection and storage of root tips
Mature rhizomes of C. amada and Z. moran were allowed to sprout on moist soil in pots for 5-10 days in the departmental green house. When the roots arose, 1-1.5 cm of root tips were collected from the sprouted rhizomes. Z. moran roots were collected between 9.30-10.00 am and C. amada roots were collected between 11.00-11.30 am respectively.
The root tips were then washed for few minutes with tap water and finally with distilled water and soaked on a filter paper. A total of 10 replicates of each plant species from different places were collected and fixed for the study.
1.3.3.2. Pre-treatment and fixation
The collected roots of Z. moran were pre-treated with saturated aqueous solution of ρ- dichloro benzene (PDB) and kept at 16 oC for 3 h. Those of C. amada were pretreated with a 1:1 mixture of saturated PDB solution and 2 mM 8-hydroxyquinoline at 4-5 °C for 4 h. The pre-treated materials were washed with distilled water for several times, soaked on a filter paper. The root tips were then fixed in freshly prepared Carnoy’s fluid (Ethyl alcohol and glacial acetic acid in 3:1 ratio), incubated for 6 hours at room temperature.
After six hours, the root tips were washed with 70 % alcohol for 3-4 times and preserved in 70 % alcohol for future use.
1.3.3.3. Hydrolysis and staining
Fixed root tips were hydrolyzed in 1 N HCl at 58 oC. Six different times (1, 2, 3, 4, 5 and 6 min) were tried for optimization of hydrolysis. Thereafter, the root tips were immersed for 2 hours in two different staining solutions, (A) Acetic acid-orcein 45% (Darlington
and Lacour 1979); (B) Acetic acid-Carmine 50% (Darlington and Lacour 1979; Harandi and Ghaffari 2001). The root tips turned black once stained.
Figure 1.1: Live specimen of Z. moran (A) and C. amada (B) in natural habitat
1.3.3.4. Slide preparation, squash and observation
The root tips stained previously were taken out on a clean microscopic slide using a brush. The root was cut gently into small pieces towards the root tip, with a razor blade and dissecting needle. Few drops of 45 % glacial acetic acid was added onto it and macerated subsequently. A cover slip was placed over the tissue trying not to get air bubbles under the cover slip. The cover slip was pressed down firmly by the thumb.
Excess of acids were soaked with filter paper. The glass was heated over the flame for few times just to soften the tissue and then squashed by pressing the slide firmly between sheets of blotting paper. With a small cork stick or pencil headed eraser the cells were spread into a monolayer. After the cells were spread uniformly, the slide was observed under the compound light microscope. Three replicates of each slide for each species were tested to avoid the potential errors of chromosome counting as well as taxonomic ambiguity.