Materials and reagents

Consumables

Eppendorf tubes, 1.5 mL (Tarsons, India)
Reagent bottles, amber 25 mL (Borosil, catalog number: 1519009)
Glass slides, 76 mm × 26 mm (Borosil, Product code 9100P02)
Coverslip, rectangular, 24 mm × 60 mm (Blue Star, India)
Aluminum foil, 72 m (Century, India)
Dissecting needle (Labkafe, India Product code: LKBI 008/1)

Plant Material

Tree plants approximately 15 years old, maintained at CSGRC’s field gene bank, of different cytotypes as diploids (MI-0014 and MI-0308), triploids (MI-0173 and MI-0799), tetraploids (K2-4X), hexaploids (ME-0126, MI-0426, and MI-0571), and decosaploid (ME-0241) were selected for this study. In the present protocol, for metaphase chromosome count, apical shoot meristematic tips were collected after 20 days of pruning.

Reagents

Sterile, double-distilled water (ddH₂O)
100% glacial acetic acid (Rankem, catalog number: A0031)
0.5 M ethylenediaminetetraacetic acid (EDTA) (GeNei, catalog number: FC43)
Ethanol (HiMedia, catalog number: MB106)
Potassium chloride (KCl) (HiMedia, catalog number: PCT0012)
2.5 (w/v) pectinase (HiMedia, catalog number: PCT1519)
2.5 (w/v) pectolyase (HiMedia, catalog number: PCT1520)
1:1 (v/v) cellulase (HiMedia, catalog number: RM3331)
1% acetocarmine (HiMedia, catalog number: PCT1304)
1% Orcein (HiMedia, catalog number: RM277)
Saturated para-dichlorobenzene (PDB) (HiMedia, catalog number: GRM6907) (see Recipes)
0.002 M 8-hydroxyquinoline (HQ) (HiMedia, catalog number: GRM7135) (see Recipes)
45% GAA (glacial acetic acid) (see Recipes)
70% ethanol (see Recipes)
3EtOH:1GAA (see Recipes)
75 mM KCl (see Recipes)
Enzyme cocktail (see Recipes)
1% aceto-orcein (see Recipes)

Equipment

Surgical blade, size 22 (Surgeon, India, REF 10122)
Personal protection equipment (Oriley, model: ORPPE6), including gloves (model: KSN30) and safety glasses (Augen, model: safety glass-SG-03)
Ice flaker (PAREX, PSW-130)
Squeeze bottle (Borosil, model: 0166024)
Highly absorbent blotting paper (Swastik, India)
Forceps, pointed, 5" (Borosil, model: LAFP8888005)
Minicooler (Tarson, model: 525060)
Incubator (ESCO, model: CCL-050B-8)
Micropipette (Eppendorf)
Freezer (Whirlpool, model: ICEMAGIC FF-350)
Stereo zoom microscope (Leica, model: Wild M8-308700)
Portable digital microscope (Medprime, model: BT-E2020) with iPad (AppleInc.)
iMac 27’’ M1 chip-macOS Monterey (Apple Inc.)
Sankalp immersion oil (Oil LV, model: 1017)
Spirit lamp (HiMedia, model: LA275)
Sealing wax (Alpha Chemika, model: AL2934)

Software

Cilika (Version 1.30), Medprime Technology Pvt. Ltd. (www.medprimetech.com), image capture software
Microsoft Excel, Microsoft
Keynote presentation software (Apple Inc. Version 10)
Floreada.io (https://floreada.io/flow-cytometry-software)

Procedure

Collections of samples and pre-treatment

Select 3–5 young healthy shoots and use needle and forceps to dissect fresh apical shoot meristematic tips, approximately from 0.5 to 1.0 cm, between 9:00 and 10:00 am. Immediately transfer the collected samples to the pre-fixative solution, i.e., 1 mL of PDB with 20 µL of HQ in a 1.5 mL Eppendorf tube (Figure 1).
Note: For pretreatment of shoot apical meristematic tips, a minimum of three (for large size tetraploid accession) and a maximum of five (small size apical tips specifically for diploid accession) samples per Eppendorf tube can be used. The sample should be transferred immediately to the pre-fixative solution, to enhance the metaphase arrest stage. PDB and HQ stock solutions should be kept separately in amber reagent bottles at room temperature (RT) and mixed properly by gently inverting five times before collection of the sample.

Figure 1. Detailed steps and daily activity for mitotic metaphase plate preparation using shoot meristematic tissues of Morus spp.

Transfer the pretreated samples to a mini cooler at 0 °C for 5 min, followed by 4 °C for 4 h.
Note: Place the mini cooler in the ice bucket before conducting sample collection for the maintenance of the mini cooler’s temperature.
Discard the pre-fixative (PDB+HQ) solution and remove young leaf primordia using pointed forceps and a needle under a stereo zoom microscope. Transfer the trimmed apical shoot tips to a strainer and wash thoroughly under running tap water for 5 min, followed by 5 mL of ddH₂O for 10 min in a watch glass (Figure 2).
Note: Removal of the young leaf primordia (usually 3–4 numbers) is useful to enhance enzymatic treatment (Step C9). Thorough washing is necessary to remove PDB+HQ residues.

Figure 2. Step-by-step flowchart of sample collection, pretreatment, fixation, staining, and squash for microscopic observation

Fixation of meristems

Transfer the washed samples to 1 mL of ice-cold 3EtOH:1GAA in a 1.5 mL Eppendorf tube and incubate for 1 h at RT (24 ± 2 °C).
Note: Prepare 3EtOH:1GAA fresh before fixation.
Discard the 3EtOH:1GAA solution and replace with freshly prepared ice-cold 3EtOH:1GAA solution. Incubate the samples at 4 °C for a minimum of two days. Replace with fresh 3EtOH:1GAA solution every 12 h.
Discard the 3EtOH:1GAA solution and wash thoroughly in 5 mL of ddH₂O for 10 min; then, store the samples in 1 mL of 70% ethanol in a 1.5 mL Eppendorf tube at 4 °C for further use.
Wash the samples in 5 mL of ddH₂O twice in a watch glass for 10 min each. Transfer the samples to 1 mL of 45% GAA in a 1.5 mL Eppendorf tube and incubate for 1 h at RT.
Wash thoroughly in 5 mL of ddH₂O for 10 min in a watch glass and remove water from the sample with a blotting paper.

Enzymatic digestion

Treat the samples with an enzyme cocktail comprised of cellulase (2%), pectinase (2.5%), and pectolyase (1%) for 4 h at 37 °C in the dark using an incubator.

Staining

Transfer enzyme-treated samples to 500 μL of 1% aceto-orcein in a 1.5 mL Eppendorf tube covered with aluminum foil (to maintain dark conditions) and incubate the sample-containing tubes for 12–14 h at RT.

Squashing (see Video 1)

Video 1. Squash technique for chromosome study of Morus spp.

Transfer one of the processed (stained) samples from the aceto-orcein stain to a glass slide.
Note: Remove the excess stain with the help of blotting paper if required.
Add two drops of 45% GAA to the sample and gently dissolve the tissue with the back side of the needle.
Gently mix the sample with the addition of one drop of acetocarmine and three drops of aceto-orcein stain; subsequently, remove the debris with a dissecting needle.
Place a coverslip over the slide. Keep the slide inside a folded blotting paper and gently press with your thumb to remove the excess stain and to spread uniformly.
Gently tap over the coverslip using the backside of the needle to obtain an optimum spread of cells as well as chromosomes.
Note: To enhance the spreading quality, continuous tapping is required until the clump of cells spread over the slide as a thin layer of cells. Ensure that no air bubbles remain.
Apply flame heat (using spirit lamp) on the bottom side of the mounted slide for 2–3 s and gently tap for precise chromosome spreading.
Seal the mounted slide with wax (optional).
Place the slide and visualize the chromosome under the microscope (Portable digital microscope, Medprime, model: BT-E2020). Any compound microscope with 40× or 100× objectives with oil immersion can be used. Microscopic images of representative accessions from each ploidy group are represented in Figure 3.

Figure 3. Metaphase plates and estimated chromosome number of different cytotypes of Morus spp. (A) diploid V1 (2n = 2x = 28), (B) triploid AR12 (2n = 3x = 42), (C) tetraploid M. laevigata L. (2n = 4x = 28), (D) hexaploid M. Serrata Roxb. (2n = 6x = 84), and (E) decosaploid M. nigra L. (2n = 22x = 308). Scale bar = 5 µm.

Flow cytometry (FCM) analysis
To confirm the ploidy level cytotypes, genome size was estimated by FCM of selected ploidy (2x, 3x, 4x, 6x, and 22x) accessions, which were identified through chromosome number count (Figure 4). A dual laser FACSCalibur^TM (BD Biosciences, United States) was used to estimate genome size with some modifications to the protocol described by Galbraith et al. (1983). In brief, young mulberry leaves (5–6 days old) of approximately 0.5 cm² were collected between 8:30 and 9:00 am. With a razor blade, the leaf sample was chopped in 2 mL of nuclear isolation buffer [hypotonic propidium iodide (50 μg/mL), trisodium citrate dihydride (3 g/L), 0.05% (v/v) Nonidet P-40, and RNase A (2 mg/mL)]; filtered (30 μm nylon mesh) nucleus suspensions were collected in tubes. The tubes were capped and kept at 37 °C for 30 min. Then, the samples were subjected to FCM analysis. Pisum sativum was used as the standard reference and measurements were made in triplicates.

Figure 4. Confirmation of different mulberry cytotypes using flow cytometry analysis. Histogram of propidium-iodine-A (PI-A) fluorescence intensity of (A) reference of Pisum sativum, (B–C) diploid, (D–E) triploid, and (F) tetraploid. (G) List of accessions studied, genome size [in mega base pairs (Mbp) and pictogram (pg)], and predicted ploidy level. (H) Histogram of PI fluorescence intensity (count vs. PI-A) of diploid (2x), triploid (3x), tetraploid (4x), hexaploid (6x), and decosaploid (22x) with reference to Pisum sativum. (I) Scatterplot (PI-W vs. PI-A) of diploid (2x), triploid (3x), tetraploid (4x), hexaploid (6x), and decosaploid (22x) nuclei, showing they are evenly spaced in respect to fluorescence and represent a well-defined series of areas that correspond to 2C, 3C, 4C, 6C, and 22C nuclei.

Data analysis

Metaphase plate images were captured (with an automated measuring scale bar) and a presentation (karyomorphological drawing) was prepared in Keynote, Apple Inc. (Version 10).
Genome size (Mbp) was calculated according to the formulae by Lysak and Dolezel (1998) with the conversion of 1 pg equal to 980 Mbp (Dolezel et al., 2003). Finally, the genome size of studied accessions was calculated using the standard reference of Pisum sativum. DNA content of the mulberry accessions ranged from 0.85 (diploid) to 8.67 pg (decosaploid) and the coefficient of variation was 3.21 (<5%; Figure 4H). The floreada.io (https://floreada.io/flow-cytometry-software) online tool was used to generate histograms and scatterplots for FCM analysis.

Notes

In step B5, change 3EtOH:1GAA solution once every 30 min for bleaching of chlorophyll.
In step B6, incubation for a minimum of two days and resuspending the sample in freshly prepared ice-cold 3EtOH:1GAA solution at 12-h-intervals is essential.
In step C9, enzymatic treatment (pectinase, cellulase, and pectolyase) for 4 h at 37 °C in the dark is recommended for karyotype analysis of higher ploidy level (2n = 3x, 4x, 6x, and 22x). For general cytological analysis and chromosome study, treating only with pectinase for 6 h at 37 °C is optimal.

Recipes

PDB
Reagent Final concentration Amount
PDB Saturated 10 g
H₂O n/a 95 mL
Total n/a 100 mL

70% ethanol
Reagent Final concentration Amount
Ethanol (absolute) 70% 70 mL
H₂O n/a 30 mL
Total n/a 100 mL

0.002 M HQ
Reagent Final concentration Amount
HQ 0.002 M 0.073 g
ddH₂O n/a 250 mL
Total n/a 250 mL

45% GAA
Reagent Final concentration Amount
GAA 45% 45 mL
ddH₂O n/a 55 mL
Total n/a 100mL

Potassium chloride (KCl)
Reagent Final concentration Amount
KCl 1 M 7.5 g
ddH₂O n/a 100 mL
Total n/a 100 mL

EtOH (3):GAA (1)
Reagent Final concentration Amount
Ethanol 3 parts 75 mL
GAA 1 part 25 mL
Total n/a 100 mL

Enzyme cocktail
Reagent Final concentration Amount
KCl 75 mM 7.5 mL
Cellulase 2% 2.0 g
Pectinase 2.5% 2.5 g
Pectolyase 1% 1.0 g
EDTA (0.5 M) 7.5 mM 1.5 mL
ddH₂O n/a 91 mL
Total n/a 100 mL

1% Aceto-orcein
Reagent Final concentration Amount
GAA 45% 45 mL
Orcein 1% 1.0 g
ddH₂O n/a 55 mL
Total n/a 100 mL

Acknowledgments

The work was supported by a grant (PIG 06004 SI) from Central Silk Board (CSB), Ministry of Textiles, GoI, Bengaluru, India. Ms. Sreya Antony thanks CSB for the Junior Research Fellowship. We are grateful to Dr. Chandish R. Ballal, Former Director, ICAR-National Bureau of Agricultural Insect Resources, Bengaluru, India for continuous encouragement and support. We thank Mr. Shreyas M. Burji, Auxochromofours Solutions Pvt. Ltd., and the team of the National Centre for Biological Sciences (NCBS), Bengaluru, India for the flow cytometry facility, and Mr. C. Ventakeshappa, CSGRC-Hosur for technical support. We would like to thank Subhankar Biswas, Department of Botany, ISc, Banaras Hindu University, Varanasi, India for constructive suggestions for video editing. We are also thankful to all three expert reviewers for their constructive suggestions/comments.

This protocol is based on the research paper (Gnanesh et al. 2023).

Competing interests

The authors declare that they have no competing interests.

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