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Journal of Stress Physiology & Biochemistry, Vol. 14, No. 4, 2018, pp. 46-53 ISSN 1997-0838 Original Text Copyright © 2018 by Rajesh Khanduji Jadhav
ORIGINAL ARTICLE
OPEN ACCESS
9
Physicocytological Invigoration of Root Meristematic Chromosomal Anomalies of Celosia argentia L. by the Genetic Potential Stress Influence of Water Hyacinth Deproteinized Foliage Whey
Rajesh Khanduji Jadhav
DG Ruparel College, University of Mumbai, Mahim, Mumbai, 400016, India
*E-Mail: [email protected]
Received December 3, 2018
In earlier studies, high concentration of DPJ from various forages inhibited germination, induced mutations in some seedlings and caused phytotoxic phenotypical changes in plants during growth. The physiological changes were absence of leaves and presence of more stipules in few pea plants. DPJ obtained from Lucerne and Eicchornia crassipes found causing chromosomal aberrations at high concentrations i.e at 0.50%, 1%, 1.5% and 2% in onion and garlic root meristems during the process of mitosis. It showed normal growth of Celosia plants by the effect of Eicchornia DPJ, when the concentrations are increased from 0.1 to 1 % level. The objective of research is to determine DPJ as mutagenic agent for effecting the cell chromosomal changes for the purpose to optimize the crops physiologically and genetically by its stress application.
During present investigation, the so called antipathogen water hyacinth DPJ potentiality changed the chromosomal response of Celosia at 1.5 %, and 2 % high concentrations. It was compared with the control. Celosia consists of the anomalous secondary growth in the root or stem. In mitotic study, mostly restitution prophases and unorientation of anaphases were found in the germinated seedling somatic cells because of the exogenous application of DPJ to the seeds.
Key words: Germination, growth, root meristem, phenols, amino acids, stress, mutation, restitution prophases, phenotypic, chromosomes
Cockscomb (Celosia argentia var cristata L.), an almost sterile hybrid between them, and eight F2 plants raised from seed from the hybrid have been studied cytologically. The hybrid possessed a chromosome number (2n=54) intermediate between those of the parents (2n=36) and 72 for cockscomb and C. argentea, respectively (William, 1954). In previous investigation, influence of DPJ was studied on leguminous plants pea and cowpea growth. DPJ enhanced the growth and the protein content of the plants. DPJ also induced the seed germination of maize, ragi and horsegram (Jadhav, 2018). After the treatment of lethal dose of DPJ of lucerne as a fertiliser when given to pea plants, there were phenotypic changes found like presence of more stipules and tallness of the stems (Jadhav, 2009a). In effect of DPJ on root growth, it was found that it induced more number of onion roots and nodulation in cowpea plants (Jadhav, 2017). At high concentration effect of DPJ in Allium cepa, there were observations of the chromosomal aberrations like micronuclei, anaphasic bridges, unorientation in metaphases, unequal daughter nuclei in telophases (Jadhav and Mungikar, 1998; Jadhav, 2009b). When the lethal dose of DPJ induced phytotoxicity in pea plants, it caused anomalous tall length of the plants with absence of leaves and only presence of stipules in some plants. DPJ contains flavonoids, phenolic compounds, lycopene etc. (Oboh, 2016). Chowdhary et al. (2002) worked on the growth of actinomycetes on DPJ, considering it as a medium.
In earlier studies, DPJ showed its potential in reducing the sizes of stomata to prevent excess transpiration with reference to drought prone area plants. DPJ consists of the potentiality to optimize the process of nitrogen fixation as because of its application to the soil if is exploited as manure (Jadhav et al., 2018; Jadhav and Gare, 2018)
A new allopolyploid species of Celosia, namely, C.
natural cross between C. argentea L. (2n=72) and C. cristata L. (2n=36), has been described and the somatic chromosome number determined as 2n=108. A somatic chromosome number of 2n=18 has been determined for another species of Celosia, namely, C. trigyna L. The basic chromosome number for the genus has been
determined as nine. Of the four species of Celosia so far studied cytologically, each represents a different level of ploidy, from diploid (2x) to dodecaploid 12x (Grant, 1961).
The present determination is the exploitation of DPJ form Eicchornia crassipes L. Cornelius et. al. (2016) showed a good antioxidant activity of Eichhornia crassipes extracts, particularly for dichloromethane and ethyl acetate, thus as an agent supplier of antioxidants. Moreover, it can be used as an alternative to help the control of E. crassipes blooms, which can affect the aquatic ecosystem. Phytochemical studies have reported compound as phenols, steroids, phenalene and humic acid commonly in plant that absorbs metals from the environment. It is antimicrobial for E. coli, Candida albicans. E. crassipes forage was found to be rich in flavonoids, amino acids, crude protein, cyanide, phosphate, silicon, calcium, potassium, organic matter and carbon. It grows in almost neutral pH, substantial concentration of dissolved oxygen, an increased rate of biological oxygen demand, a substantial concentration of nitrates and very low salinity and alkalinity. Investigation of various amino acids in Eichhornia leaf extract indicates that it consists of phytohormones, as tryptophan and methionine are precursors of auxins, gibberellins and cytokinins (Lara-Serrano et al., 2016; Nyananyo et al., 2007). P. Jayanthi et al. (2011) investigated the proteins in Eichhornia leaves by ninhydrin and biurets method and phenols. The presence of phenolic content indicates the presence of salicylic acid, a plant hormone which is a compound of phenol in Eichhornia, favoured the seed germination and seedling growth of Celosia at appropriate concentrations of DPJ.
In order to evaluate the potential of DPJ as a mutagen to optimize the crop characteristics phenotypically and genotypically its effect on mitosis in root meristem is the present objective (Kothekar et al., 1995; Satpute and Ghagre, 2009; Esra et al., 2018). Celosia seeds were germinated by the treatment of increasing concentrations of water hyacinth DPJ. The results obtained were increase in the length and weight of the seedlings as per the increase in DPJ concentration (Jadhav, 2018). During present
whiteii, collected in Malaya and originating from a
investigation, the DPJ dose application to seeds exogenously was given at high concentration beyond 1 to 2% to evaluate anomalies.
MATERIALS AND METHODS
Preparation of Eicchornia DPJ
By the process of green crop fractionation, the forage of water hyacinth was pulped and squeezed to obtain the juice. The juice is heated to 90°C, filtered in order to obtain the leaf protein concentrate (LPC) on filter paper. The curd like extract after heating forms the supernatant is called as deproteinized juice (DPJ) or leafy whey, as the proteins gets coagulated by seperation. The DPJ is dried in oven and various concentrations were prepared by weighing it in grams to treat for the seed germination of Celosia by paper towel method. High concentrations of DPJ were 1, 1.5 to 2 %. The seeds were soaked for 24 hours in DPJ made up of Eicchornia crasipes L. to have the effect on germination of seeds. The cytological studies were followed by using the root tips of Celosia argentia seedlings after the germination.
Cytological Investigations of Celosia Roots Grown In Vitro
Ninety (90) seeds of Celosia argentia L. (Century mixed) were germinated on moist filter paper. Primary roots produced in vitro were also made into permanent slides and analysed for cellular behaviours, such as Mitotic index and chromosome count. The experiments were conducted using a light microscope images. Treated and Control seeds were placed in folded whatman paper, lined with moist tissue paper. The healthy root tips 1 to 1.5 cm were cut which were pre-treated with 1 %, 1.5 % and 2 % of DPJ. The root
alcohol until ready for viewing. The meristems were hydrolysed in 1N HCl (hydrochloric acid) for 15 minutes and squashed on a glass slide. They were then stained in 2 % acetocarmine for 1 hour and viewed under light microscope. Frequency of each phases of cell division was counted and the Mitotic index (MI) estimated. Five replicate slides were prepared for each treatment (Abubakar et al, 2015).
RESULTS AND DISCUSSION
During the amino acid analysis in the Celosia foliage itself , it revealed high contents of the essential amino acids with methionine as the limiting amino acid. Presence of methionine in extract reveals its hormonal availability (Aodele and Olajide, 2011). In earlier experiment, there was no influence of DPJ at low concentration i.e at 0.12 % during the seedling growth of Celosia. When the concentrations increased at 0.50 % , 0.75 % and 1.00 %, there was found promotion in length and weight of the seedlings of Celosia plants as per the enhancing concentrations of DPJ by soaking treatment. Table 1 illustrates that comparatively during cell division, majority of abnormalities were found in the anaphases by unequal segregation or unorientation of the chromosomes. Anaphases were also had bridges and laggards. There was incomplete arrest of cell division at the stage of prophase because there was restitution in it at 15.21 % of the mitotic index. Many of the chromosomes were having the stickiness. Majority of prophases were suffered from scattering, disintegration, fragmentation and granulation. Balloon nuclei were found in case of metaphases and during the stage of telophase. The telophases were less in number. Comparatively the telophases were very small in sizes. The process of cell division was declined after the stage of anaphase, because of impact of higher concentration of DPJ. Therefore the table indicates that there was no complete mitotic arrest because of the application of DPJ at 1.5 % level. DPJ when treated at 2% level, there was arrest of mitosis as prophases suffered from aberrations like scattering and condensation in many nuclei. Hence the total chromosomal aberrations by calculating mitotic index because of DPJ was 93.45 %. In earlier findings it was already investigated that high concentration of lucerne DPJ i.e at 2% level, caused mitotic arrest in many nuclei. Jadhav and mestry (2018) founded that Allium cepa DPJ inhibits the enzyme protease of the fungi Trichoderma viride. Haggag et al. (2017) found antifungal activity of Eichornia against six pathogenic fungi viz. Aspergillus flavus, A. niger, Alternaria alternata, Colletotrichum gloeosporioides, Candida albicans, and Fusarium solani. Therefore, very few normal nuclei were found by Eicchornia DPJ
meristems were then fixed in 1:3 (v/v) glacial acetic acid and alcohol solution for 24 hours to fix cell division. These isolated meristems were preserved in 70 %
influence. However the investigation proves that there was the mutagenic influence of DPJ on seedlings of Celosia plants. In order to study the change in chromosomal number of Celosia or other genus because of DPJ, research is advisable. DPJ influence
varies as per the species of plants used. The study prevails that the relevant concentration of DPJ in the present investigation for the purpose of mutational studies was of 1.5 % for giving the treatment.
Table 1. Mutagenic influence of Eichhornia DPJ on seed germination at various concentrations and study of
chromosomal aberrations in the somatic cell nuclei of Celosia argentia.L from root meristems.
No. DPJ Treatment (%) Chromosomal abnormalities (CA) Mitotic Index (%)
1 2 Restitution of prophases 15.21
2 2 Scattering or disintegration of prophases 8.69
3 2 Fragments or granulated nuclei 13.04
4 2 Condensation of the nuclei 8.69
5 1.5 Baloon nuclei of metaphases 6.52
6 1.5 Stickiness in chromosomes 17.39
7 1.5 Unorientation or unequal segregation 23.91
Total (M.I.) Percent of abnormalities 93.45
Table 2.
Percent Mitotic Index of each stages by DPJ influence on Celosia somatic cells.
No. Chromosomal Abberations in mitotic stages Mitotic Index, (%)
1 Prophase 50
2 Metaphase 39.13
3 Anaphase 26.08
4 Telophase 15.21
5 Total 67.39
Chromosomal abberations found in Celosia root tips by DPJ influence
■ Mitotic Index (%)
Figure 1. Graphical presentation of the chromosomal aberrations found by the influence of the DPJ on somatic cell division of Celosia argentia.
Figure 2. Illustration of the somatic chromosomal anomalies (CA) of Celosia root tips. (A) Stickiness of the chromosomes, Normal Prophase and metaphase. (B) Telophase having long distance among the daughter cells, prophase and disturbed anaphases. Normal telophase having small daughter nuclei. (C) Disturbed metaphase and unequal segregation of anaphases. (D) Restitution of prophase and unorientation of balloon shaped metaphase. (E) Interphases and restitution of prophase. (F) Sticking of two metaphases together. (G) Late and earlier metaphases. (H) Disturbed Anaphase with bridges and laggards. (I) Disintegrated and scattered prophase, unequal segregation in anaphases and presence of metaphase. (J) Unequal segregation in anaphases, restitution, condensation and fragmentation of prophases and earlier metaphase. Labelled figures (pictures) P=prophase, M=Metaphase, A=Anaphase, T= teophase.
Figure 3. Graphical illustration of chromosomal anomalies in different mitotic stages.
Mitotic Index (%) of different stages
■
■
— ■ 1
Prophase Metaphase Anaphase Telophase
U Mitotic Index (%)
Graphical presentation in figure 1 illustrates the higher abnormalities scores of chromosomes were found during the anaphase stage i.e of unequal segregation of chromosomes towards two opposite poles. Figures 2 (A) to (J) indicates that there were chromosomal aberrations. The nuclei found of cockscomb somatic cells were broader in size comparatively with Allium cepa and A. sativum. During slide preparation, due to excessive chromosomes, there was breakage and dispersal of many chromosomes found on the slide.
Figure 2 (E) very clearly reveals presence of normal interphases. Normal prophases are shown in figures 2
The restitution and disintegration of the prophase stages are depicted in figures 2 (D), (E) and (I). Abnormal metaphases are shown in figures 2 (C), (D) and (F). There are two normal prophases and one normal anaphase in figure 2 (B). Abnormalities in anaphase was found dual. Those laggards and unorientations among chromosomes. Unequal segregation in anaphase is shown in figures 2 (C), (I) and (J). Figure (H) shows the anaphasic bridges and laggards. In figure 2 (B), the telophase is consisting of long abnormal distance among the two daughter nuclei.
Mitotic activities observed in the cells of the treated plants and the control are presented in Table 2. Estimated mitotic index values showed that DPJ concentration at 2% plants which had the highest percentage of abnormalities illustrated in table 1. Mitotic
index values obtained illustrated in table 2 determines that in all the untreated cells of the control was 80.36, higher than that of the total of treated. Treatements total had the least value (67.39). The most occurring mitotic stages is the prophase (50) followed by the metaphase (39.13) and anaphase (26.08). Least stages of telophases were found as these stages were not got divided from anaphase (Figure 3). Reason of least telophases is unequal segregations of chromosomes and bridges i. e the biphasic anomalies in anaphasic stages. There was decreased mitotic index of the treated plants compared with the control. It was reported that there were inhibitory effects of water hyacinth DPJ on the mitotic index of Celosia. This means that DPJ can have a genotoxic and mutagenic effects. C. argentea, the plant in which aberrations induced, contains saponins, cyclic-peptides, phenols, fatty acids, amino acids, minerals, of which saponins are the main pharmacological active agents and might be the promising target for further studies due to their bioactivities and the mechanisms of saponins have been performed. Anti-mitosis in the moroidin (6)/celogentin families in Semen Celosiae has been reported (Tang et al., 2016).
Similar effect of DPJ from lucerne was observed on onion and garlic root tips. It is opined that ATP deficiency caused by excessive constituents of DPJ which comes during heating of leaf juice at 90°C after filtration. It may be one of the reasons for the decrease in the mitotic index and demands for ATP of dividing cells are much higher as compared to non proliferating
(A) and (C), metaphases in figures 2 (A), (G) and (I). Normal anaphase and telophase shown in figure 2 (B).
cells. DPJ is enriched with all nutrients especially salts, hormones, DNA and RNA. DPJ also contains non protein nitrogen and very few amino acids despite it is deproteinised. These Chromosomal aberrations were
analyzed in metaphase chromosomes detected within the cell population of only a single primary root out of the whole seedling grown by DPJ treated seeds after germination, which is a strong evidence for its spontaneous nature (Nikolova, 2015).
CONCLUSION
The present study invigorated that, the high concentration of Eichhornia DPJ acted as the mutagenic agent on Celosia argentia weed/ornamental plants by disturbing the cell cycle. However, study reveals that the mutagen DPJ have the potentiality to induce the complex chromosomal changes in Celosia plants at 1.5% as this plant bear the anomalous secondary growth of stem. The stress of DPJ by its application have the efficacy in physiological changes and the nature of chromosomes in plants because of its chemistry and hormonal content. Investigation of change in chromosomal number in plants due to DPJ will be the next objective of the research. Other commercial seeds will be taken into consideration for genetical research to make the crops more optimistic like production of disease resistant variety. Chromosomal aberrations play a vital role in evolution as they generate variation in a natural population. Aberrations result in altered linkage relationships and this has been exploited for breeding experiments.
ACKNOWLEDGEMENTS
The Author thanks to the authorities of the D. G. Ruparel College, Mahim, Mumbai for providing the laboratory facilities for completion of this minor research project funded by University of Mumbai, Mumbai. India.
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