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.
Assam, Nagaland and Arunachal. The rhizome of this species is off white in color, with strong pungent smell, smaller in size and is less fibrous than the common ginger. The immature rhizomes are reddish white and the scale leaves are much darker. Flowers are solitary, born in a racemose spike which is dense cone like usually develop during the vegetative growth of the plant. Corolla are light yellow with a black-yellow spotted labellum. Flowers are bisexual, zygomorphic and epigynous. Fruits absent.
1.4.2. Identification and maintenance
The collected plants of C. amada and Z. moran were identified following the taxonomic identification keys.
Curcuma amada:
Key to the Kingdom: Presence of chlorophyll, autotrophic, whole body is divided into
stem, root and leaves. --- Plantae
Key to the Division: Plants with flowers and seeds, ovules enclosed within the ovary --- Angiosperms Key to the Sub-division: Leaves simple with parallel venation, Flowers mostly
trimerous, one cotyledon. --- Monocotyledons
Key to the Order: Inflorescence racemose, flowers bisexual, zygomorphic, creeping horizontal or tuberous rhizomes. --- Zingiberales
Key to the Family: Flowers zygomorphic, epigynous, only one fertile stamen, other stamens transform into staminodes. --- Zingiberaceae Key to the Sub-family: All parts aromatic, leaves distichously arranged in two rows, sometimes tufted or single, sheaths open on side opposite lamina, lateral staminoids usually petaloid or represented by a teeth at the base of the labellum, aromatic oil present
--- Zingiberoideae Key to the Genus: Inflorescence central appear with leaves with a terminal plume of sterile bracts, peduncle enclosed within leaf sheaths, several flowers forming a spike, each flower or each cluster of flowers is subtended by 2 bracts, reddish or pinkish white
in color, nectaries present. --- Curcuma
Key to the Species: Ovary inferior, rhizomes with sessile branched tubers, possess
characteristic flavor of raw mango. --- amada.
Floral diagram:
Floral formula:
Zingiber moran:
Key to the Kingdom: Presence of chlorophyll, autotrophic, whole body is divided into
stem, root and leaves. ---Plantae
Key to the Division: Plants with flowers and seeds, ovules enclosed within the ovary --- Angiosperms Key to the Sub-division: Leaves simple with parallel venation, Flowers mostly
trimerous, one cotyledon. --- Monocotyledons
Key to the Order: Inflorescence racemose, flowers bisexual, zygomorphic, creeping horizontal or tuberous rhizomes. --- Zingiberales Key to the Family: Flowers zygomorphic, epigynous, only one fertile stamen, other stamens transform into staminodes. --- Zingiberaceae Key to the Sub-family: All parts aromatic, leaves in two rows, nectaries present.
--- Zingiberoideae Key to the Genus: Flowers solitary, each cluster of flowers is subtended by a bract.
--- Zingiber Key to the Species: Ovary inferior, petals yellow, bracts green, labellum is yellow-black, spotted, rhizome with characteristic odor, dirty or off-white in color, particularly cultivated by the ‘Moran’ tribe of NE India. --- moran
% +♂Br2P3+3 A2+4 G(3)
Floral diagram:
Floral formula:
Hence the systematic position of the two species is identified as:
Kingdom: Plantae Kingdom: Plantae
Division: Angiosperms Division: Angiosperms
Sub division: Monocotyledons Sub division: Monocotyledons
Order: Zingiberales Order: Zingiberales
Family: Zingiberaceae Family: Zingiberaceae
Genus: Curcuma Genus: Zingiber
Species: amada Species: moran
Collected plants were replanted under green house conditions in pots prepared with alluvial soil and sand mixture (3:1) and maintained as live specimen. Figure 1.2 depicts the Z. moran plant maintained in greenhouse. These were also maintained as herbarium specimen for future reference (Figure 1.3).
% +♂P3+3 A2+4 G(3)
Figure 1.2: Live specimen of Z. moran (A) in green house;
An inflorescence of Z. moran (B)
Figure 1.3: Herbarium specimen of C. amada (A) and Z. moran (B)
B A
B
A
1.4.3. Chromosome counts 1.4.3.1. Collection of root tips
To check the ploidy level of the collected species, chromosome counts of C. amada and Z. moran were carried out. The best time of the day to collect root tips for karyological study is 10 am - 12 pm where there are more cell metaphases. However the time was optimized for both the plants under study and accordingly growing root tips were collected between 9.30- 10.00 am for C. amada and 11.00 – 11.30 am for Z. moran. Both the periods were found to give adequate numbers of metaphases in respective plants.
However, the midday period is widely followed in the field of cytology for the family Zingiberaceae (Lim 1972b; Newman 1990; Nair and Sasikumar 2009). Midday is known to be at a peak of cell division in many plants and thus will yield the highest number of metaphases when fixed for cytological observation. Late-morning period was also preferred by some workers (Chen 1992; Augsonkitt et al. 2004).
1.4.3.2. Pretreatment
Pre-treatment is a necessary step in cytological studies of members of Zingiberaceae.
Different workers have used various pre-treatments and staining chemicals in their studies (Chen 1992; West and Cowley 1993; Rai et al. 1997; Das et al. 1998; Joseph et al.
1999; Nair and Sasikumar 2009). This helps to block the mitosis cycle at metaphase stage by inhibiting spindle fiber formation revealing more number of cells in metaphase. PDB and 1-bromonapthalene (MBN) were found better in treating the Zingiberaceae plants by Chen (1992). However, in this study it was found that PDB alone was effective in treating Z. moran and a mixture of PDB and 8-hydroxyquinolene (OQ) gave a slightly higher percentage of metaphase cells for C. amada. Our result matches with the findings of Rai et al. (1997) and Das et al. (1998). Literature on cytological study says that PDB and OQ are preferred for the pre-treatment of Curcuma and Zingiber species.
1.4.3.3. Hydrolysis and staining
Hydrolysis of the root tips is carried out to soften the root tissue. The roots of monocots are harder and larger size and therefore, it is an important step to soften the pretreated roots for a specified period in 1N HCl. In the present study, the time was optimized as 5
min for C. amada and 3 min for Z. moran respectively at 58 oC. Later, the roots were allowed to cool down at room temperature and subjected to dye (2% aceto-orcein or aceto carmine) in a test tube and heated over a flame for few seconds. The heating for a few seconds in the acid-dye mixture intensified the staining of the chromosomes and gave a clearer general picture of the chromosome structure. Both the stains tested, were found to give better results in the two plants under study. However, Feulgen has also proved to be an effective in staining Zingiberaceae (Lim 1972a; Newman 1990; West and Cowley 1993). Orcein is another stain giving fruitful results in many members of Zingiberaceae (Nair and Sasikumar 2009).
1.4.3.4. Slide preparation, squash and observation
The preparation of a good slide depends on the proper handling of the root material, cutting, maceration and squashing. Care was taken so that the tip of the root is not hurt.
Adding of the acid onto the root tip slowly macerate it and also decolorize the cytoplasmic material leaving only the chromosomes stained. The squashing and spreading of the cells uniformly is a crucial step which results in proper visualization of the slide. The slides revealed well-spread metaphases under low power (10x) and high power (40x) magnifications in compound light microscope. Some cells were found to be in anaphase and late prophase stages also.
Out of 10 varieties studied, C. amada confirmed the chromosome number of 2n = 42 where as Z. moran revealed the chromosome number of 2n=22 without any polyploidy (Figure 1.4). The slides showing the chromosome counts are shown in figure 1.3. The occurrence of different ploidy levels in Curcuma was highlighted in early cytological studies (Suguira 1931; Suguira 1936; Raghavan and Venkatasubban 1943;
Venkatasubban 1946; Chakravorty 1948; Sharma and Bhattacharya 1959). Considering the widely accepted basic chromosome number n=21 (Ramachandran 1961; Prana 1977;
Islam 2004), this chromosomal variation roughly corresponds to three euploid levels (2n, 3n and 4n) plus several aneuploids. The ploidy level of C. amada matches with the findings of other workers (Das et al. 1999). However Z. moran’s chromosome number is a new count. Chromosome structures of the species studied are metacentric.
Figure 1.4: Slides showing chromosome number of C. amada, 2n=42 (A);
Z. moran 2n=22 (B)