2019 Volume 84 Issue 4 Pages 313-317
Piper nigrum is an important age-old herbal plant of the family Piperaceae. Chromosomal analysis a part of genomic research to conserve plant genetic resources has confronted difficulties in this particular species with conventional methods primarily due to its very small chromosome size and rich cytoplasmic contents. Different chromosome number from 2n=52 to 128 have been reported in this species. A reassessment of chromosome analysis not attempted earlier with aerial roots and enzymatic maceration and air drying (EMA) method has been standardized in this species. Well scattered cytoplasm free metaphase plates stained with Giemsa revealed 2n=52 very small chromosomes. Total chromosome length attains to 75.36±011 µm. Most chromosome pairs except one ranged from 0.99 to 1.81 µm. Karyotype formula is determined as 6M+26m+20sm and accordingly, idiogram is constructed. Fluorochrome banding with two contrasting nucleic acid dye DAPI and CMA provided positive signals for both of the stains on the very small chromosomes. The standardized EMA method and fluorochrome banding is repeatable and will help to evaluate other important species and genus within this family for the conservation of plant genetic resources.
Black pepper or P. nigrum L., one of the three most important cultivated pipers, belongs to the family Piperaceae. The species ranked first as a spice in cookery for its flavor and aroma and is crowned as “Black Gold” and “King of Spices” (Gorgani et al. 2017). In traditional medicine ‘Piper is an age-old herbal plant of Ayurveda’ (Ahmad et al. 2012). Many reports on morphological, biochemical, molecular and biotechnological aspects of P. nigram are available (Narayanan 2000, Ravindran 2000, Nair and Gupta 2003, Nazeem et al. 2005, Parthasarathy et al. 2007, Shivashankar 2014) but the species has confronted extreme difficulties in the field of chromosomal studies. Chromosomal analysis in economically important crops has benefited the conservation of plant genetic resources for any crop improvement programs. Two basic chromosome numbers x=12 and 13 have been reported for the genus Piper. Since Johansen (1931) initiated chromosome analysis in the genus, a number of workers have attempted chromosome analysis in P. nigrum and reported different chromosome numbers. Janaki Ammal (1955) reported 2n=128 chromosomes. Jose and Sharma (1984) reported 2n=104, chromosomes. Sharma and Bhattacharyya (1959) reported 2n=48, Dasgupta and Datta (1976) reported 2n=36 and 60 chromosomes. Whereas 2n=52 chromosomes were reported by Mathew (1958), Martin and Gregory (1962), Rahiman and Nair (1986). Even Nair et al. (1993) reported 2n=78 chromosome. All authors have used conventional orcein staining for determination of chromosome number and reported that chromosomes of P. nigrum are very small in size and cells contain dense cytoplasm. No chromosome morphology was described in this species. However, it is agreed that chromosome analysis still provides foundational genomic information in a very cost-effective manner and “comparison of the molecular, genetic and cytological classification of chromosomes remains a highly relevant task” (Muravenko et al. 2009). It appears that the species did not receive adequate attention it deserves. A reassessment of chromosome analysis in this particular species with the application of the EMA method not attempted earlier was felt. EMA method was introduced for rice chromosomes by Kurata and Omura (1978) and has been applied in many plant species. The method is the basics of molecular cytogenetics and suitable for all types of plant chromosomes especially for cells having rich cellular contents and small chromosomes (Schweizer 1976, Kondo and Hizume 1982, Hizume 1991, Fukui 1996, Yamamoto 2012, Jha et al. 2015). EMA method followed by Giemsa staining not only helps in counting the actual chromosome number but also clears chromosome morphology through the removal of cell wall and cytoplasmic derbies. It was further revealed that earlier authors have used roots induced from vegetative cuttings due to problems of seed germination. Therefore, the present communication details the standardization of EMA based chromosome preparation method from aerial roots in P. nigrum along with the karyotype analysis through Giemsa and fluorochrome banding for the first time.
P. nigrum aerial roots induced normally in nodal zones of a potted plant under moist and humid conditions were used for chromosome analysis (Fig. 1). A minimum of 20 healthy roots was collected in between 11.30 to 12 am and pre-treated separately with saturated paradichlorobenzene (PDB) and 0.5% colchicine solution for 4–5 h at 14–16°C. Acetic methanol (1 : 3) was used as a fixative and kept the roots in this solution for overnight and preserved in −20°C. Our earlier protocol (Jha et al. 2015) for EMA method with required standardization of enzyme maceration time from 55–100 min at 37°C was followed chromosome staining time with Giemsa, 4′-6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA) was also optimized. Giemsa stained slides were de-stained in 70% methanol and air-dried prior to fluorescent staining. Basic steps of Kondo and Hizume (1982) were followed for fluorescent staining. However, 0.1 µg mL−1 and 0.2 µg mL−1 of DAPI solution for 15–30 min and 0.1 mg mL−1 of CMA solution for 60–100 min were tried for fluorescent staining. Slides were mounted in non-fluorescent glycerol and CMA slides were kept for maturation at 4°C for more than 48 h.
For counting of chromosome number and karyotype analysis a minimum of 50 well scattered Giemsa stained metaphase plates were examined. Similarly, 20–25 DAPI and CMA stained plates were also studied for verification of chromosome number and fluorescence response. A Carl Zeiss Axio Lab A1 microscope having a bright field, filter cassettes of DAPI and CMA, CCD camera fitted with a computer was used to capture all the photographs in the present studies. An Axiovision L. E4 software was used for the analytical part. For karyometric analysis standard karyological parameters were used (Jha et al. 2017).
Means and standard deviations were analyzed for all measured parameters. One-way analysis of variance (ANOVA) was performed to detect significant differences (p≤0.05) in the mean (Sokal and Rohlf 1995). Duncan’s multiple range test (DMRT) was used for post hoc analyses using SPSS v 16.0 statistical package (SPSS Inc. IBM, Chicago).
Processing of aerial roots (Fig. 1B) with PDB and colchicine yielded many metaphase (Fig. 2) and pro-metaphase plates. It revealed that both pretreating chemicals can be used for chromosome preparation. An important observation is that the aerial roots can be regularly used as an alternative tissue for chromosome preparation along with the induced roots from vegetative cuttings in P. nigrum. Enzymatic digestion time in EMA method and Giemsa staining time are two critical steps for the removal of the cell wall and rich cytoplasmic contents and staining of such small chromosomes which is required for the correct determination of chromosome number and analysis of chromosome morphology. Both the steps were optimized and large number of cytoplasm free metaphase and pro-metaphase plates from aerial roots of P. nigrum were obtained when root tips were treated in enzyme solution for 95–100 min at 37°C and stained with 1.5% Giemsa solution in phosphate buffer for 30 min. Chromosomes of P. nigrum are very small in size and for confirming the diploid number more than 50 well scattered metaphase plates were studied. More than 95% cell counts revealed 2n=52 chromosomes (Fig. 2A, B). For karyometric analysis five well-scattered metaphase plates with distinct chromosome morphology were used. Out of 26 pairs of small chromosomes, length of 25 pairs ranged from 0.99 to 1.81 µm and only one pair was 2 µm long. Detail chromosome morphometric data is presented in Table 1. Total chromosome length of 52 chromosomes appears from the table is 75.36 ±0.11 µm and karyotype formulae is 6M+26m+20sm. Idiogram of the species is presented in Fig. 2C. The present chromosome count 2n=52 confirms the reports of Mathew (1958), Martin and Gregory (1962) and Rahiman and Nair (1986).
Chromosome number (2n) | Length of longest chromosome (µm) | Length of shortest chromosome (µm) | Total chromosome length (µm) (Mean±S.D.) | Total form percent (TF %) (Mean±S.D.) | Karyotype formulae (2n) | ||
---|---|---|---|---|---|---|---|
Absolute (Mean±S.D.) | Relative (Mean±S.D.) | Absolute (Mean±S.D.) | Relative (Mean±S.D.) | ||||
52 | 2.00±0.02 | 2.65±0.03 | 0.99±0.02 | 1.31±0.03 | 75.36±0.11 | 38.73±0.11 | 6M+26m+20Sm |
Fluorescent banding with CMA and DAPI which generally targets GC- and AT-rich constitutive heterochromatin regions on chromosomes and generate positive and negative signals have advanced cytogenetic research for many plant species (Schweizer 1976, Kondo and Hizume 1982, Hizume 1991, Moscone et al. 1996, Yamamoto 2012, Schwarzacher 2016). Not only they offer additional help in counting the chromosome number but also mark the chromosomes throughout the karyotype (Kondo and Hizume 1982, Hizume 1991, Guerra 2008). The concentration of fluorochrome dyes and staining time has been standardized in P. nigrum. DAPI staining was better with 0.2 µg mL−1 for 30 min while 0.1 mg mL−1 CMA took nearly 100 min time for differential staining in the same metaphase plates. It is reported that plants having small chromosomes face difficulties with fluorescent banding due to insufficient resolving power of light microscope and relatively low copy of repetitive sequences. However, for the first time P. nigrum revealed positive signals for two contrasting CMA and DAPI stains. The present chromosomal analysis was carried out on prometaphase and metaphase chromosomes of P. nigrum and has obtained at least 12 and 18 positive CMA and DAPI signals (Fig. 3). The standardized EMA method, Giemsa staining and fluorochrome banding are repeatable and well documented. The present observations not only conserve the genetic features of P. nigrum but also are useful in characterisation and conservation of intra and inter genetic diversity within the economically important genus Piper.
The author dedicates the paper to his teacher cum mentor Late Prof. A. K. Sharma, a renowned internationally reputed Indian cytogeneticist for sharing his own experience on this particular species. TBJ also acknowledges Dr. S. Dutta, Principal, and Dr. P. Roy Head, Dept. of Botany Maulana Azad College, Kolkata for providing all basic facilities and Dr. P.S. Saha for his all required technical support.