Indian Journal of Agricultural Research

  • Chief EditorV. Geethalakshmi

  • Print ISSN 0367-8245

  • Online ISSN 0976-058X

  • NAAS Rating 5.60

  • SJR 0.293

Frequency :
Bi-monthly (February, April, June, August, October and December)
Indexing Services :
BIOSIS Preview, ISI Citation Index, Biological Abstracts, Elsevier (Scopus and Embase), AGRICOLA, Google Scholar, CrossRef, CAB Abstracting Journals, Chemical Abstracts, Indian Science Abstracts, EBSCO Indexing Services, Index Copernicus
Submit

Full Research Article

Morphophysiological Parameters of Growth and Development of Seedlings of Gleditsia triacanthos L. in the Early Stages of Ontogenesis under Conditions of Changing Lighting Intensity

Pavel Krylov1,*, P.A. Kuzmin2, K.A. Melnik3
1Laboratory of Genomic and Postgenomic Technologies, Federal State Budget Scientific Institution “Federal Scientific Centre of Agroecology, Complex Melioration and Protective Afforestation of the Russian Academy of Sciences” 400062, Volgograd, pr-t Universitetskij, 97, Russia.
2Laboratory of Molecular Breeding, Federal State Budget Scientific Institution “Federal Scientific Centre of Agroecology, Complex Melioration and Protective Afforestation of the Russian Academy of Sciences” 400062, Volgograd, pr-t Universitetskij, 97, Russia.
3Laboratory Bioecology of Woody Plants, Federal State Budget Scientific Institution “Federal Scientific Centre of Agroecology, Complex Melioration and Protective Afforestation of the Russian Academy of Sciences” 400062, Volgograd, pr-t Universitetskij, 97, Russia.

ABSTRACT

Background: Gleditsia triacanthos L. is a valuable taxon for agroforestry. This species is promising for the creation of protective plantations and has a wide range of economically valuable traits, in particular, drought tolerance. In this regard, the aim of the work was to evaluate morphophysiological parameters of growth and development of G. triacanthos seedlings at early stages of ontogenesis under conditions of changing light intensity. 

Methods: To achieve this goal, seeds of G. triacanthos were sown with subsequent selection of individuals for the study possessing homogeneity of genetic structure. Analysis of close relatedness of individuals of G. triacanthos showed low intraspecific diversity by Nei (0.27 to 0.62). To evaluate the development of seedlings of G. triacanthos at early stages of ontogenesis, morphophysiological methods were used. 

Result: Under unfavorable conditions due to low light, the growth and development of the embryonic root and seedling formation was slow. However, all subphases of G. triacanthos seed organogenesis were passed in 98% of individuals, thus forming seedlings under standard criteria. Accumulation of total chlorophyll content in unfavorable conditions was 3 times less compared to individuals in favorable conditions. At the same time, there was a tendency to increase the sum of chlorophylls in the leaves of G. triacanthos in each experimental group at 30 days (p<0.05). As a result, it was revealed that light intensity is a limiting parameter for accelerated growth and development of G. triacanthos seedlings.

INTRODUCTION

The “response-factor” system is one of the key traits for selecting woody shrub plants for the breeding process and solving agroforestry tasks. Southern territories of Russia are characterized by difficult climatic conditions for the growth of woody plants (Manaenkov et al., 2020; Kulik et al., 2023; Belyakov et al., 2024). Search and selection of new promising species of woody and shrubby plants is necessary to increase species diversity (Tyshchenko et al., 2023), having high forest and biological productivity and resistance to a wide range of negative environmental factors (Simpson, 2019).
       
One of such promising genera for creation of protective plantations of various functional purposes is G. triacanthos (Dizaji et al., 2016). The most widespread species is G. triacanthos, which grows in southern chernozems, dark chestnut and light chestnut soils with solonetz complexes (Sergio et al., 2020; Semenyutina et al., 2021). Also G. triacanthos, has a wide range of economically valuable traits (Redwan, 2019; Melnik and Khuzhakhmetova, 2022).
       
In connection with the above-mentioned it is urgent to search and select G. triacanthos individuals with high productivity and resistance to unfavorable habitat factors at early stages of ontogenesis for further breeding and planting in dry-steppe zones (Belyaev et al., 2022; Carta et al., 2022). The main selection criteria are morphological and physiological-biochemical parameters, particularly the pigments of the photosynthetic apparatus in the early stages of ontogenesis and genetic structure. The genetic structure of woody plants can be determined by using DNA markers represented by ISSRs (Ramzan et al., 2021; Saitwal et al., 2022; Abd-Dada et al., 2023). These markers identify the presence of loci that may be associated with a particular economically valuable trait in woody plants (Novikova et al., 2012). Since the G. triacanthos genome has not been sequenced (Belyaev et al., 2023), ISSR markers, which are non-specific multilocus markers (Luo et al., 2018; Tantasawat et al., 2021), are best suited to detect these loci.
       
The aim of the work was to evaluate morphophysiological parameters of growth and development of G. triacanthos seedlings at early stages of ontogenesis under conditions of changing light intensity.

MATERIALS AND METHODS

G. triacanthos seeds were collected from individuals of good vital mid-age ontogenetic condition located on the basis of cluster dendrology collections (#34:34:000000:122 - woody plant nursery of the Federal Scientific Center of Agroecology of the Russian Academy of Sciences). The research was conducted on the basis of the Federal Research Center for Agroecology of the Russian Academy of Sciences in the laboratories of molecular breeding, as well as bioecology of woody plants from March 2022 to February 2024. Collected seeds of G. triacanthos seeds were scarified before germination by immersion in concentrated sulfuric acid for 120 minutes, followed by rinsing in water for a day to destroy seed coating (Lorca et al., 2019; Balakina et al., 2021).
       
Swollen seeds of G. Triacanthos were placed in a seed germination tray for 36 h in a climate chamber at 26°C. After emergence, the seeds were placed in the soil. Next, two groups were formed which included 20 plants each.
       
To study the effect of illumination on the early stages of ontogenesis of seedlings of G. triacanthos. G. triacanthos, the conditions were modeled by changing the length of daylight hours. In the control group the optimal photoperiodism regime was set (illumination 50...260 lx), in the experimental group the duration of the light day was reduced to 7 hours (illumination 2...16 lx). Analyzed parameters were taken after 7, 15 and 30 days and material for genetic analysis was collected and stored at -20°C.
       
Evaluation of morphological parameters of ontogenesis at early stages of G. triacanthos was carried out by measuring plant height (cm) and leaf length (cm). The measurement was performed for all leaves of each individual and the mean value was indicated, which were used in statistical processing. Metabolites were quantified in leaves using a Dualex Scientific+ plant analyzer (Force-A, France) after 7, 15 and 30 days. For the counted plant, the analyses were carried out in three biological repetitions and the mean value was indicated, which were used in statistical processing. Measurement of chlorophylls (μg/cm2 raw weight), flavonoids (μg/cm2 raw weight), anthocyanins (μg/cm2 raw weight) and nitrogen balance index (NBI) in leaf epidermis of woody plants.
       
Genomic DNA was extracted from 50 mg of leaf plates using a modified CTAB method with the addition of 0.2% b-mercaptoethanol (Shestibratov et al., 2021). The concentration and quality (A260/A280) of DNA in the samples were assessed using a Qubit 4.0. fluorimeter (Thermo Fisher Scientific Inc., USA) and a SpectrostarNano spectrophotometer (BMG Labtech, Germany), respectively. For ISSR detection we used primers showing high amplifiability, synthesized by Evrogen CJSC, Russia (Table 1).
 

Table 1: Name and sequence of the ISSR samples.


       
The ISSR-PCR-based amplification was conducted using kit qPCRmix-HS (cat: #PK145L) (CJSC Evrogen, Russia). The ISSR-PCR-based amplification was performed using kit qPCRmix-HS (cat: #PK145L, CJSC Evrogen, Russia) according to the manufacturer’s instructions. The Applied Biosystems QuantStudio 5 (Thermo Fisher Scientific, USA) was used to amplify genomic DNA with ISSR markers. The conditions of ISSR-PCR amplifications included initial denaturation at 95°C for 10 min followed by 40 cycles at 95°C for 30 sec, annealing temperature for the various primers at 55°C for 30 sec (extension) and final extension at 72°C for 4 min.
       
The 2% agarose gel electrophoresis with TAE buffer was used to separate the results of the ISSR-PCR reactions (Al-Khayri et al., 2022). The size of DNA bands on the gel was determined using a 100+bp DNA ladder (cat: #NL002, CJSC Evrogen, Russia). Ethidium bromide (Helicon, Russia) was used to stain the agarose gel, which was then visualized using a UV illuminator (VilberLourmat, France). The frequencies of polymorphisms and the number of bands produced by each primer were calculated individually.
       
The frequency of polymorphisms and the number of bands produced by each primer were calculated individually. ISSR loci were scored as present “1” or absent “0”, each considered as independent. Data were compiled into a 0/1 binary data matrix using MS Excel. The generated binary data were used to calculate the polymorphism of ISSR loci in the POPGENE version 1.31 (Louati et al., 2019). Quantitative data were processed using Statistica 12.0 software (StatSoftInc., USA) with calculation of the indices accepted to characterize nonparametric samples in biomedical research: median, 1st quartile, 3rd quartile (Me [Q1 ÷ Q3]). The Kraskell-Wallis test for multiple groups and the Mann-Whitney test for two independent samples (p<0.05) were applied to prove the reliability of differences.

RESULTS AND DISCUSSION

Analysis of intraspecific genetic diversity in G. triacanthos seedlings by five ISSR markers showed homogeneity of the seed population, which confirms their common biotype. Based on the obtained data of amplicon spectra, binary matrices were compiled for further mathematical processing in POPGENE to estimate the intraspecific genetic diversity in G. triacanthos seedlings (Fig 1).
 

Fig 1: Example of electrophoregram of amplification products of seedlings G. triacanthos with ISSR primer UBC 841, Std molecular weight marker (100-1000 bp).


       
Genetic parameters of seedlings of G. triacanthos are given in Table 2. The effective number of alleles for the primers used ranged from 1.23 to 1.76, indicating the close relatedness of the oospores under study. It was revealed that seedlings of G. triacanthos seedlings had the highest effective number of alleles at the UBC 841 locus, which shows greater individuality of the genetic structure compared to other primers. One of the key parameters for assessing the closely relatedness of individuals of G. triacanthos was applied by Ney genetic distance estimation, which showed low values indicating genetic homogeneity. The Shannon index also showed low values ranging from 0.27 to 0.62, indicating low intraspecific diversity. As a result, loci UBC 811 and UBC 841 were identified whose parameter values were 1.5-2 times higher than those of UBC 808, UBC 835 and UBC 836.
 

Table 2: Indicators of genetic diversity of seedlings G. triacanthos.


       
The results of the analysis show homogeneity of the genetic structure, which will increase the representativeness of the data obtained for other morphophysiological parameters under experimental conditions.
 
Study of morphological parameters of seedlings G. triacanthos
 
Under unfavorable conditions, seeds of G. triacanthos seeds developed passively due to low light conditions. The stage of germinal root growth and seedling formation was slow. However, all subphases of organogenesis of seeds of G. triacanthos passed 98% of the individuals, thus forming seedlings under standard criteria. The height of plants was several times higher than in favorable conditions, this is explained by the fact that seedlings tried to find an additional light source (Fig 2A).
 

Fig 2: Morphological parameters of seedlings G. triacanthos.


       
The greatest number and length of leaves were recorded in the seedlings that grew under favorable conditions (p<0.05). This variant did not need anything, thus developed its biomass (maximum leaf length) and underwent growth and development much more intensively (p<0.05) (Fig 2B). This is in agreement with the results of growing seedlings Gleditsia caspica L. (Nourmohammadi et al., 2019).
 
Study of the content of pigments of the photosynthetic system of seedlings G. triacanthos
 
The content of chlorophyll sums in G. triacanthos leaves differed between seedlings in favorable and unfavorable conditions 7 days after sprouting and after 15 and 30 days no significant differences were found (Fig 3A). After 7 days under favorable conditions, the content of chlorophyll sum in G. triacanthos seedlings was 3 times higher compared to individuals under unfavorable conditions (p<0.05). At the same time, there was a tendency to increase the sum of chlorophylls in G. triacanthos leaves in each experimental group by 30 days (p<0.05).
 

Fig 3: The content in the plant leaves G. triacanthos.


       
There were no statistically significant differences in flavonoid content in G. triacanthos individuals under favorable and unfavorable conditions throughout the experiment (Fig 3B). This may be primarily due to the fact that flavonoids are responsible for the functioning of the plant immune system (Middleton, 2009), also under laboratory conditions, no disease or leaf lamina lesions were observed.
       
Under unfavorable conditions, G. triacanthos individuals had 2 and 4 times higher anthocyanins content after 7 and 15 days than seedlings growing under favorable conditions (p<0.05) (Fig 3C). It was found that there was no difference in anthocyanins content after 30 days between the experimental groups (p>0.05). This indicates an important role of anthocyanins in the early stages of ontogenesis in the formation of adaptive potential (Cappellini et al., 2021).
       
In G. triacanthos individuals growing under favorable conditions, the value of nitrogen balance index on 7 days was 4 times higher (p<0.05) than in unfavorable conditions and on 15 days the difference was reduced by 2 times (p<0.05)(Fig 3D). After 30 days, no statistically significant differences were observed. During the whole experiment, there was an increase in the index value of nitrogen balance index (p<0.05).

CONCLUSION

Data on the growth and development of G. triacanthos seedlings at the early stages of ontogenesis grown under conditions of light deficiency were obtained. Genetically homogeneous seedlings of G. triacanthos formed a specific adaptive response to the conditions of light deficiency. According to the results of pigment system analysis, a pronounced reaction of G. triacanthos seedlings to an important factor of growth and development - illumination - was revealed by changes in the content of the sum of chlorophylls and anthocyanins. The study of morphological parameters of G. triacanthos seedlings in different conditions showed that the lack of light is a limiting parameter for accelerated growth and development of seedlings in laboratory conditions. Individuals of G. triacanthos in unfavorable conditions undergo all stages of ontogenesis with lower intensity than individuals in favorable conditions.

ACKNOWLEDGEMENT

The research was carried out on the topic of the State Assignment of the Federal Scientific Center for Agroecology, Integrated Land Reclamation and Protective Afforestation of the Russian Academy of Sciences: No. 122020100449-3 “Search for breeding valuable genetic material for the creation of new genotypes of tree and shrub species by molecular breeding methods” and No. 121041200195-4 “Formation of multifunctional cluster dendrological expositions and their renovation into bioresource artificial and landscaped landscape spaces of recreational type in sparsely wooded regions of Russia”.

CONFLICT OF INTEREST

All authors declare that they have no conflicts of interest.

REFERENCES


  1. Abd-Dada H., Bouda S., Khachtib, Y., Bella Y.A., Haddioui, A. (2023). Use of ISSR markers to assess the genetic diversity of an endemic plant of Morocco (Euphorbia resinifera O. Berg). Journal, Genetic Engineering and Biotechnology. 21(1): 91. DOI: 10.1186/s43141-023-00543-4.

  2. Al-Khayri, J.M., Mahdy, E.M.B., Taha, H.S.A., Eldomiaty, A.S., Abd- Elfattah, M.A., Abdel Latef, A.A.H., Rezk, A.A., Shehata, W.F., et al. (2022). Genetic and morphological diversity assessment of five kalanchoe genotypes by SCoT, ISSR and RAPD-PCR markers. Plants (Basel, Switzerland). 11(13): 1722. DOI: 10.3390/plants11131722.

  3. Balakina, A.A., Nefedieva, E.E., Larikova, Yu, S. (2021). Investigation of the structure and composition of the seed cell of Gleditsia and some changes in its structure during swelling. Agrarian Bulletin of the Urals. 03 (206): 46-52. DOI: 10.32 417/1997-4868-2021-206-03-46-52.

  4. Belyaev, A.I., Semenyutina, A.V., Khuzhakhmetova, A.Sh., Semenyutina, V.A. (2022). Analysis of bioresource collections on climatic rhythms and phenological processes. Ecological Engineering and Environmental Technology. 23(3):87-94. DOI: 10.129 12/27197050/147152.

  5. Belyaev, A.I., Krylov, P.A., Pugacheva, A.M., Derevshikova, L.V. (2023) Analysis of the presence of the genomes of wood and shrubs plants used in agro-forest-melioration in the southern regions of Russia. Proceedings of lower Volga agro-university complex: science and higher education. 2(70): 30-42. DOI: 10.32786/2071-9485-2023-02-03.

  6. Belyakov, A.M., Koshelev, A.V., Nazarova, M.V., Egorov, A.V. (2024). On the formation of ecologically balanced agrolandscapes in the dry-steppe zone of chestnut soils. Indian Journal of Agricultural Research. 58(1): 106-112. DOI: 10.18805/ IJARe.AF-801.

  7. Cappellini, F., Marinelli, A., Toccaceli, M., Tonelli, C., Petroni, K. (2021). Anthocyanins: From mechanisms of regulation in plants to health benefits in foods. Front. Plant Sci. 12: 748049. DOI: 10.3389/fpls.2021.748049.

  8. Carta, A., Mattana, E., Dickie, J., Vandelook, F. (2022). Correlated evolution of seed mass and genome size varies among life forms in flowering plants. Seed Science Research. 32(1). DOI: 10.1017/S0960258522000071.

  9. Dizaji, F.E., Kafi, M., Khalighi, A., Jari, K.S. (2016). Phytoremediation of lead and cadmium by thornless honey loccust trees’ (Gleditsia triacanthos L. var. inermis) in contaminated soil near the Tehran-Karaj highway. Indian Journal of Agricultural Research. 50(6): 579-583. DOI: 10.18805/ ijare.v50i6.6676.

  10. Kulik, K.N., Belyaev, A.I., Pugacheva, A.M. (2023). The role of protective afforestation in combating drought and desertification of agricultural landscapes. Arid Ecosystems. 1(94): 4-14.

  11. Lorca, A., Ferreras, À., Funes, G. (2019). Seed size and seedling ontogenetic stage as modulators of damage tolerance after simulated herbivory in a woody exotic species. Australian Journal of Botany 67(2):159-164. DOI: 10.107 1/BT18093.

  12. Louati, M., Ucarli, C., Arikan, B., Ghada, B., Hannachi, A.S., Turgut- Kara, N. (2019). Genetic, morphological and biochemical diversity of argan tree (Argania spinosa L.) (Sapotaceae) in Tunisia. Plants (Basel, Switzerland). 8(9): 319. DOI: 10.3390/plants8090319.

  13. Luo, C., Chen, D., Cheng, X., Liu, H., Li, Y., Huang, C. (2018). SSR analysis of genetic relationship and classification in chrysanthemum germplasm collection. Horticultural Plant Journal. 4(2): 73-82. DOI: 10.1016/j. hpj.2018.01.003.

  14. Manaenkov, A.S., Rybashlykova, L.P. (2020). Increasing the efficiency of plant-cover restoration in the modern focus of deflation on pastures of the northwestern Caspian region. Arid Ecosystems. 10(4): 358-367. DOI: 10.1134/S20790961 20040149.

  15. Melnik, K.A., Khuzhakhmetova, A. Sh. (2022). Features of fruiting of introduced representatives of the generic Gleditsia complex in the age aspect. Proceedings of the Nizhnevolzhsky agrouniversitetskiy complex: Science and Higher Professional Education. 4(68): 184-193. DOI: 10.32786/ 2071-9485-2022-04-21.

  16. Middleton, E. (2009). Effect of plant flavonoids on immune and inflammatory cell function. Advances in experimental medicine and biology, 439:175-182. DOI: 10.1007/978- 1-4615-5335-9_13

  17. Nourmohammadi, K., Kartoolinejad, D., Naghdi, R., Baskin, C.C. (2019). Effects of dormancy-breaking methods on germination of the water impermeable seeds of Gleditsia caspica (Fabaceae) and seedling growth. Folia Oecologica. 46(2): 115-126. DOI: 10.2478/foecol-2019-0014.

  18. Novikova, A.A., Sheikina, O.V., Novikov, P.S., Doronina, G.U. (2012). Estimation of the ISSR-markers application for systematization and genetic certification of Genus Rhododendron. Scientific Journal of KubSAU. 82: 79-89.

  19. Ramzan, F., Kim, H.T., Younis, A., Ramzan, Y., Lim, K.B. (2021). Genetic assessment of the effects of self-fertilization in a Lilium L. hybrids using molecular cytogenetic methods (FISH and ISSR). Saudi Journal of Biological Sciences. 28(3): 1770-1778. DOI: 10.1016/j.sjbs.2020.12.019.

  20. Redwan, O. (2019). Germination of the seeds of Gleditsia triacanthos L. and the effect of some agricultural communities on the growth and development of their seedlings. Biological Sciences Series. 41(6): 11-26.

  21. Saitwal, Y.S., Musmade, A.M., Kale, A.A., Supe, V.S., Joshi, V.R., Ranpise, S.A., Mehetre, S.S. (2022). Characterization of Annona Genotypes by ISSR and SSR Markers. Bhartiya Krishi Anusandhan Patrika. 37(1): 43-49. DOI: 10.18805/ BKAP494.

  22. Semenyutina, A.V., Khuzhakhmetova, A.Sh., Dolgikh A.A., Sapronov, V.V. (2021). Scientific basis for enriching the dendroflora of sparsely wooded regions with economically valuable plants. World Ecology Journal. 12(2): 35-51. DOI: 10.257 26/v1064-3614-5462-o.

  23. Sergio J., Yohana, G., Romina, D. (2020). Structure of the forests of Gleditsia triacanthos as a function of age (Valle de La Sala, Tucumán, Argentina). Austral Ecology. 30: 251-259. DOI: 10.25260/EA.20.30.2.0.1083.

  24. Shestibratov, K.A., Mescherova, E.N., Krutovsky, K.V., Baranov, O.Y., Kiryanov, P.S., Panteleev, S.V., Mozharovskaya, L.V., Padutov, V.E. (2021). Structure and phylogeny of the curly birch chloroplast genome. Frontiers in Genetics. 12(FEB): 625764. DOI: 10.3389/fgene.2021.625764.

  25. Simpson, M. (2019). Evolution and diversity of woody and seed plants. Plant Systematics. pp. 131-165. DOI: 10.1016/ B978-0-12-812628-8.50005-5.

  26. Tantasawat, P.A., Poolsawat, O., Kativat, C., Arsakit, K., Papan, P., Chueakhunthod, W., Pookhamsak, P. (2021). Association of ISSR and ISSR-RGA markers with powdery mildew resistance gene in mungbean. Legume Research. 44(10): 1164-1171. DOI: 10.18805/LR-606.

  27. Tyshchenko, A., Tyshchenko, O., Konovalova, V., Fundirat, K., Piliarska, O. (2023). Methods of determining the drought resistance of plants. InterConf. DOI: 10.51582/interconf. 19-20.05.2023.030.
Reviewed By

Download Article
In this Article
    Contact us
    Follow us

    Editorial Board

    View all (0)