Abstract: Background and Objective: Botanical gardens are conservatories where living plants are conserved for scientific, academic and awareness programs. Assessments of existing cytotypic diversity at inter and intraspecific levels provide a real pen picture of the genetic diversity of germplasm present in any ex situ conservatory like botanical garden. Therefore, the present investigation was undertaken for the assessment of cytotypic diversity in targeted taxa. Materials and Methods: A total number of 51 species of economically important angiosperms which are growing in the botanical garden of Botanical Survey of India, Central Regional Center and Allahabad were investigated for evaluation of existing cytotypic diversity. Meiotic preparations were made as per the standard squash preparation technique. Results: Among the worked-out species 47 species were represented by a single cytotype and the remaining four by two cytotypes. Besides, new cytotypes were recorded for 07 species of angiosperms which are possibly new to science. Conclusion: The findings of the present investigation are important in terms of the assessment of genetic diversity and conservational aspects of the aforesaid botanical garden. The identified unique germplasm of the concerned species will be utilized by the researchers who are working on genetic improvement and gene identification programs.
INTRODUCTION
Knowledge of available cytotype diversity in any life form provides clues on ongoing evolutionary processes as well as provide information regarding unique germplasm viz., aneuploids, euploids, polyploids, etc. Such germplasm is very important and utilized by researchers in chromosome identification, gene identification, crop improvement programs, etc. Hence their identification, information about their availability and place of availability are very essential. Botanical gardens are sites where plants are gathered together for systematic studies. At present globally 2500 botanic gardens are existing which cumulatively conserve 6 million accessions of living plants, representing around 80000 taxa1,2. According to Dosman3 botanical gardens are favourite sites for taxonomic and systematic studies as plants of different geographical and agroclimatic zones are available under one roof. Although, botanical gardens have great potential to contribute to different streams of biological sciences, inadequate knowledge of existing intraspecific and infraspecific genetic diversity limits their exploration4.
The Botanical Garden of Botanical Survey of India, Central Regional Center, Allahabad (BSI, CRC, Allahabad) is situated at 181 m Altitude, 250 28'North and 810 51'Longitude and covers about 2.5 ha. It nurtures 663 species having medicinal, economically important, ornamental, rare and threatened plants belonging to 118 families5. The garden comprises many sections including aquatics, arboretum, bambusetum, gymnosperms, medicinal plants, net house, ornamental plants, plant introduction, RET species and rosary. Despite of vast gathering of unique germplasm of different species till the year 2019, no effort was made to explore the cytotypic diversity in available germplasm. Hence, in the light of above-mentioned facts, the present investigation was undertaken to explore the existing cytotypic diversity in targeted plant species.
MATERIALS AND METHODS
Study area: The present investigation was carried out between 2020-2021 at the botanical garden of Botanical Survey of India, Central Regional Center, Allahabad.
Study design: In the case of trees and shrubs, the cytological investigation was performed on an individual plant basis whereas in herbaceous it was on a population basis. For cytological investigations requisite plant materials were collected on each plant basis or population basis which depends on the habit of the species. The chromosomal count in each case was made through male meiotic preparation for which appropriate sized buds were fixed in Carnoy’s fixative (6 Ethanol: 3 Chloroform: 1 Acetic acid v/v) for 24 hrs then the material was transferred into 70% ethanol and stored in the refrigerator at 4°C. The meiotic squash preparations were made in 2% acetocarmine adopting standard methodologies. Slides were observed under the Nikon Eclipse 200 microscope and chromosome counts were made.
RESULTS AND DISCUSSION
In the present investigation 51 species of angiosperms belonging to 37 genera and 20 families, conserved in the Botanical Garden of BSI, CRC, Allahabad were cytologically analyzed. In Table 1 the information on available cytotypes, their ploidy level, earlier reported chromosome count, etc. were summarized. Among them, 28 and 23 species were analyzed on a population and individual plant basis, respectively. As per Chromosome Counts Database(http://ccdb.tau.ac.il) and Chromosome Atlas of Flowering Plants of Indian Subcontinent6 current findings showed close resemblance with the findings of earlier workers. Forty seven species were represented by a single cytotype, the remaining four viz., Cassia fistula (2n = 26, 28), Rauvolfia tetraphylla (2n = 44, 66), Solanum nigrum (2n = 24, 72) and S. villosum (2n = 24, 72) by two cytotypes. Review of the literature indicates that for C. fistula three cytotypes (2n = 24, 26, 28) were reported so far of which 2n = 28 is found most common. The cytotype 2n = 24 and 2n = 26 was found rare and reported by a few workers6-8. The observations on Rauvolfia tetraphylla support the findings of earlier workers9-11. For S. nigrum and S. villosum two cytotypes (2Xi, 6x) were observed which showed consonance with the findings of Melo et al.12, Kumar and Pushpangdhan13.
Bogunić et al.14 opined that genomic and cytotypes diversity is possibly a major cause of reproductive isolation and finally leads to speciation. Discovery of new cytotypes is always challenging but it is desirable because they will be utilized as experimental material for a better understanding of ongoing evolutionary processes15. In the present investigation, new cytotypes were recorded for seven species viz. Allium tuberosum (2n = 28), Chlorophytum nepalense (2n = 26), Chlorophytum tuberosum (2n = 30), Crotalaria spectabilis (2n = 26), Ocimum basilicum (2n = 78), Sansevieria cylindrica (2n = 38) and Sansevieria trifasciata (2n = 56) which are possibly new to science6,7.
Table 1: | Study pattern and cytological observations in different taxa |
Species | Family | Study pattern | Observed chromosome No. (2n) | Ploidy | Reported chromosome number (By earlier workers) |
Achyranthes aspera L. (x = 7) | Amaranthaceae | Population basis | 28 | 4x | 28, 42,48, 56, 96 |
Allium tuberosum Rottler ex Spreng. (x = 7) | Amaryllidaceae | Population basis | 28* | 4x | 24,31,33,32,62 |
Aloe vera (L.) Burm. F. (x = 7) | Xanthorrhoeaceae | Population basis | 14 | 2x | 14, 21,38 |
Antigonon leptopus Hook. and Arn. (x = 7) | Polygonaceae | Population basis | 42 | 6x | 14,40, 42, 44,48 |
Asparagus racemosus Willd (x = 11) | Asparagaceae | Population basis | 22 | 2x | 20, 22, 40 |
Azadirachta indica A. Juss. (x = 14) | Meliaceae | Individual plant | 28 | 2x | 28, 30 |
Basella alba L. (Green) (x = 11,12) | Basellaceae | Individual plant | 44 | 4x | 44, 48 |
Bauhinia purpurea L. (x = 14) | Leguminosae | Individual plant | 28 | 2x | 28 |
Bauhinia variegata L. (x = 14) | Leguminosae | Individual plant | 28 | 2x | 28 |
Boerhavia diffusa L. (x = 13) | Nyctaginaceae | Population basis | 56 | Aneuploid | 26, 52, 116 |
Caesalpinia pulcherrima (L.) Sw. (x = 11, 12) | Leguminosae | Individual plant | 28 | Aneuploid | 22,24,28 |
Cassia fistula L. (x = 6, 7, 8; x2 = 13) | Leguminosae | Individual plant | 26, 28 | 2 x2; 2x | 24,26, 28 |
Chlorophytum comosum (Thunb.) Jacques (x = 7) | Asparagaceae | Population basis | 28 | 4x | 28 |
C. nepalense (Lindl.) Baker (x = 7, 8) | Asparagaceae | Population basis | 26* | Aneuploid | 28,40,42, 56 |
C. tuberosum (Roxb.) Baker (x = 7) | Asparagaceae | Population basis | 30* | Aneuploid | 16 |
Crotalaria spectabilis Roth (x = 8) | Leguminosae | Population basis | 26* | Aneuploid | 16,24 |
Datura stramonium L. (x = 12) | Solanaceae | Population basis | 24 | 2x | 24, 25,36, 48 |
Delphinium ajacis L. (x = 8) | Ranunculaceae | Individual Plant basis | 16 | 2x | 16, 24 |
Gymnema sylvestre (Retz.) R. Br. ex Sm. (x = 11) | Apocynaceae | Individual plant | 22 | 2x | 22 |
Haworthiopsis limifolia (Marloth) G. D. Rowley (x = 7) | Xanthorrhoeaceae | Population basis | 28 | 4x | 14,21,28 |
Helicteres isora L. (x = 9) | Malvaceae | Individual plant | 18 | 2x | 18,20,24,38 |
Justicia simplex D. Don (x = 9) | Acanthaceae | Individual plant | 18 | 2x | 18, 36 |
Justicia adhatoda L. (x = 7, 8, 9; x2 = 17 ) | Acanthaceae | Population basis | 34 | 2x | 34,40,46, 50 |
Lantana camara L. (x = 11) | Verbenaceae | Population basis | 44 | 4x | 22, 32, 33,36, 44, 55, 66 |
Lantana montevidensis (Spreng.) Briq. (x = 11, 12) | Verbenaceae | Individual plant | 48 | 4x | 22, 36, 48 |
Ocimum basilicum L. (x = 8) | Lamiaceae | Population basis | 78* | Aneuploid | 30, 48, 52, 54, 56, 72,74 |
Oroxylum indicum (L.) Curz. (x = 14, 15) | Bignoniaceae | Individual plant | 28 | 2x | 28, 30, 38 |
Papaver rhoeas L. (x = 7) | Papaveraceae | Population basis | 14 | 2x | 14, 15,16,18, 21, 28,30, 42 |
Phlomoides superba (Royle) Kamelin & Makhm (x = 11) | Lamiaceae | Individual plant | 22 | 2x | 22 |
Physalis minima L. (x = 12) | Solanaceae | Population basis | 48 | 4x | 24, 48, 72 |
Pongamia pinnata (L.) Pierre (x = 10, 11) | Leguminosae | Individual plant basis | 22 | 2x | 20, 22 |
Rauvolfia serpentina (L.) Benth. ex Kurz (x = 10, 11) | Apocynaceae | Population basis | 22 | 2x | 20, 22, 44 |
Rauvolfia tetraphylla L. (x = 11) | Apocynaceae | Population basis | 44, 66 | 4x, 6x | 44, 55, 66, 68, 88 |
Sansevieria cylindrica Bojer ex Hook (x = 14*a) | Asparagaceae | Individual plant | 38* | Aneuploid | 28, 36, 42 |
Sansevieria trifasciata Prain (x = 9*a) | Asparagaceae | Individual plant | 56* | Aneuploid | 18, 40, 84, 92, 102, 103, 104, 112,119, 120 |
Sansevieria zeylanica (L.) Willd. (x = 20,21) | Asparagaceae | Individual plant | 40 | 2x | 40, 42 |
Santalum album L. (x = 10) | Santalaceae | Individual plant | 20 | 2x | 10,20,40 |
Saraca asoca (Roxb.) Willd.(x = 12) | Leguminosae | Individual plant | 24 | 2x | 24 |
Senna alata (L.) Roxb. (x = 6, 7, 8) | Leguminosae | Individual plant | 28 | 4x | 24, 28 |
Senna obtusifolia (L.) H.S. Irwin & Barneby (x = 6, 7, 8) | Leguminosae | Population basis | 28 | 4x | 24, 28, 52, 56 |
Senna tora L. (x = 6, 7, 8) | Leguminosae | Population basis | 28 | 4x | 26, 28, 52,56 |
Senna sulfurea (Collad.) H. S. Irwin & Barneby (x = 6, 7, 8) | Leguminosae | Individual plant (2) | 28 | 4x | 28, 56 |
Solanum diphyllum L. (x = 12) | Solanaceae | Population basis | 24 | 2x | 24 |
Solanum nigrum L. (x = 12) | Solanaceae | Population basis | 24, 72 | 2x, 6x | 24, 36, 48, 60, 64, 72, 96, 144 |
Solanum villosum L. (x = 12) | Solanaceae | Population basis | 24,72 | 2x, 6x | 24, 48, 50, 72 |
Solanum virginianum (x = 12) | Solanaceae | Population basis | 24 | 2x | 24 |
Tamarindus indica L. (x = 12) | Leguminosae | Individual plant | 24 | 2x | 24, 26, 28 |
Tinospora cordifolia (Willd.) Miers ex Hook.f. and Thoms (x = 12) | Menispermaceae | Population basis | 26 | Aneuploid | 24, 26 |
Uraria picta (Jacq.) DC. (x = 8) | Leguminosae | Individual plant (2) | 16 | 2x | 16, 22 |
Urena lobata L. (x = 7) | Malvaceae | Population basis | 28 | 4x | 14,28, 56 |
Withania somnifera (L.) Dunal (x = 12) | Solanaceae | Population basis | 48 | 4x | 24, 48, 72 |
*: New chromosome count (Cytotype) for particular species, x: Basic chromosome number, x2: Secondary basic chromosome number and *a: Basic chromosome count assumed on the basis of lowest 2n number |
A total number of 26 species are identified as diploid, 15 as tetraploid, 04 as hexaploid and 09 as aneuploidy individual-containing species (Table 1). de Queiroz16 and Meudt et al.17 opined that ploidy change is very crucial for the speciation and diversification of plants. According to maximum workers change in the ploidy level of plants affects their qualitative and quantitative attributes and individuals having these changes are important for genetic improvement programs, the study of evolutionary trends, etc. In the case of Boerhavia diffusa, Caesalpinia pulcherrima, Chlorophytum nepalense, C. tuberosum, Crotalaria spectabilis, Ocimum basilicum, Sansevieria cylindrica, S. trifasciata, Tinospora cordifolia aneuploidy individuals were also observed (Table 1). Aneuploids generally formed by the gain or loss of chromosomes from the normal set of chromosomes, are very crucial for locating a linkage group and a gene in a particular chromosome18. Hence, there is a need to conserve these identified elite genotypes including aneuploids18.
Lastly, cytological techniques have a number of limitations viz. time factor, requirement of advanced microscope facility and trend professions but the findings of present investigation prove the potential of the mentioned technique in assessment of existing genetic diversity in any germplasm and identification of the elite genotypes of particular species. The findings were also important in terms of research including genetic improvement program for economically important species, academics and formulation of conservation strategies. Keeping the mentioned facts and requirement of assessment of genetic diversity of any germplasm collection in mind similar types of investigations are recommended for the botanical gardens of India.
CONCLUSION
The findings of the present investigation on the one hand demonstrated the importance of cytological studies in the assessment of genetic variability in germplasm holdings of ex situ conservatories like botanical gardens on the other hand demonstrated the urgent need to start similar types of studies. Besides these, this type of studies will definitely provide help to the researchers who want to work on unique germplasm of economically or scientifically important plant species as search, identification and characterization of unique germplasm of any plant species is a tedious task.
SIGNIFICANCE STATEMENT
The present investigation was undertaken for the assessment of existing cytotypic diversity in germplasm collection of fifty-one targeted plant species. The key findings includes identification of rare cytotype for C. fistula (2n = 26) and new cytotype for Allium tuberosum (2n = 28), Chlorophytum nepalense (2n = 26), Chlorophytum tuberosum (2n = 30), Crotalaria spectabilis (2n = 26), Ocimum basilicum (2n = 78), Sansevieria cylindrica (2n = 38) and Sansevieria trifasciata (2n = 56) which are possibly new to science. Besides these polyploid and aneuploid individuals were identified for 19 and 09 species of angiosperms respectively which are very important in terms of gene identification and genetic improvement programs of economically important species.
ACKNOWLEDGMENTS
Present work carried out under Annual Action Plan Project of Botanical Survey of India. The author is grateful to Director, BSI, Kolkata, and HoO BSI, CRC, Allahabad for providing all necessary facilities and support for executing the present research work.