Effects of Ascorbic Citric and Humic Acids On Maiz

Published Online First: September, 2024
https://doi.org/10.21123/bsj.2024.10020
P-ISSN: 2078-8665 - E-ISSN: 2411-7986 Baghdad Science Journal
Effects of Ascorbic, Citric, and Humic Acids on Maize Stem and Leaf
Anatomy
Jazran Jard Kadhum1 , Jalal Hameed Hamza*2 , Muazaz Azeez Hasan2,3 , Maythem
Al-Amery 4 William Serson 5 , Mahmoud F Seleiman 6 , Martin Battaglia7 , Hail
Z Rihan 8
1
Ministry of Education, Babylon Education, Vocational Education, Babylon, Iraq.
2
Department of Field Crops, College of Agricultural Engineering Sciences, University of Baghdad, Baghdad, Iraq.
3
Department of Biology, College of Pure Sciences-Ibn Al-Haiytham, University of Baghdad, Baghdad, Iraq.
4
Department of Biology, College of Science for Women, University of Baghdad, Baghdad, Iraq.
5
Pennsylvania State University. 2809 Saucon Valley Rd, Center Valley, PA 18034, United States.
6
Department of Plant Production, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia.
7
Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VA 24061, United States
8
School of Biological and Marine Sciences, Faculty of Science and Engineering, University of Plymouth, United
Kingdom.
*Corresponding Author.
Received 24/10/2023, Revised 31/05/2023, Accepted 02/06/2024, Published Online First 20/09/2024
© 2022 The Author(s). Published by College of Science for Women, University of Baghdad.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Abstract
Anatomical changes in internal tissue of stem and leaf when seed and plant treated with acids to enhance
growth and development in maize was studied during the spring seasons of 2019 and 2020. Randomized
complete block design was used with three replications. Main plots received foliar nutrition treatments,
including ascorbic acid (AA), citric acid (CA), and humic acid (HA) at concentrations of 100 mg L -1,
alongside HA at 1 ml L-1, with distilled water as the control. Sub-plots underwent corresponding treatments
for seed soaking. Results indicated variations in vascular bundle size among treatments, with foliar CA
treatment showing superior results in both years, as well as seed soaking in CA and HA. Interaction effects
were observed, notably in 2019 with the combination of foliar CA and seed soaking with distilled water,
and in 2020 with HA. Effects on leaf epidermis were minimal, with slight distortions in stomatal shapes
observed with AA and CA treatments compared to the control. AA and HA treatments led to larger ordinary
epidermal cells with straighter cell walls than the control, along with an increase in cork and silica cell size
in treated plants. This study contributes to understanding anatomical modifications in maize leaves and
stems during the growing season, shedding light on the potential impacts of acid treatments on plant
physiology.
Keywords: Foliar nutrition, Growth regulators, Plant anatomy, Seed soaking, Zea mays L.
Introduction
Maize holds significant importance as a crop cultivation. However, the cultivation of grain corn in
cultivated for its seed, silage feed, and its role in this region is confronted with numerous
human and animal nutrition. In Iraq, there has been a environmental challenges, ultimately resulting in
noticeable increase in the area dedicated to maize diminished productivity1,2. One critical factor
contributing to this is the selection of inappropriate tolerance by improving photosynthetic pigments,

maize varieties, which can exacerbate issues such as osmo-protectants contents and antioxidant system,
moisture accumulation within the grain. Such which lead to protect maize plant from damaging of
accumulation poses a threat to the safe ripening of drought stress specially in the early stages of growth.
the crop at harvest time. Thus, the careful selection AA is a cofactor for control the phytohormones,
of maize varieties tailored to the specific enzymes, cell growth and development and can be
more effective in mitigating the harmful effects of
environmental conditions of Iraq is paramount to
water deficit27.
ensure optimal productivity and harvest quality.
Citric acid improves abiotic stress tolerance through
Many challenges have emerged in the area, including better osmoregulation, induces antioxidant defense
the soil degradation and desertification, the scarcity systems, promotes increased chlorophyll content and
of water, the harshness of climatic conditions, and relieving heavy metal stress28. Citric acid is a low-
global climate changes due to global warming. These molecular-weight organic acid exuded by the plant
have been exacerbated in the absence of proper roots. Organic anions derived from this acid compete
agricultural planning, but they can be confronted for phosphorus adsorption sites in clay minerals. CA
through the use of techniques and technology in the use led to increase P availability of corn plants29.
application of modern agriculture, which is the basis Growth, photosynthetic pigments, and biomass
through which these challenges can be faced, and this increased by enhancing antioxidant enzyme activity
represents the research hypothesis3,4. with the application of B. vietnamiensis and CA30.
Soaking seeds in growth regulators before planting Citric acid foliar led to modification of pH and
can be a beneficial practice in mitigating the negative induction of macrobiotic activity of the rhizosphere
effects of environmental stress on the growth, or the capacity to form complexes with metallic ions
physiological and biochemical responses of crops3,5. or the mobilization of phosphorus. CA has an effect
Environmental stress factors such as salinity6, high on the physiology leaves, especially in the increase
temperatures7,8, and drought9 can pose significant of soluble protein and proteolytic activity31.
challenges to plant growth and overall crop Humic acid (HA) is recognized as an important class
productivity. Growth regulators typically affect of antioxidant compounds commonly found in
germination time, steering to preferable growth and natural water and soil. These compounds play a
amended yield10, exceptionally in plants under the significant role in participating in extensive redox
stress11,12. reactions within ecosystems. It is worth noting that
Farmers keep trying to avoid chemical fertilizer by HA can account for up to 80% of dissolved organics
using some sustainable practices such as applications in natural water sources32,33. Humic acid as a foliar or
of biofertilizer or sea weed13-15, plant extracts and pre-sowing seed treatment significantly increased the
plant regulators16-18 which led to enhance plant biomass, chlorophyll pigments and proline
germination, growth and yield19-21 significantly after contents. Foliar spray was better in improving plant
seed soaking or spraying during vegetative stages biomass, chlorophyll contents, accumulation of
even under stresses of drought and salt22,23. nutrients, however, in contrast, seed pre-treatment
Foliar ascorbic acid alleviated the negative effects of was more effective in altering leaf proline. HA led to
Cadmium stress in maize and improved enhance salinity tolerance through HA-induced
photosynthetic processes, osmolytes, and antioxidant increase in plant biomass, content of chlorophyll and
defense systems24. Ascorbate is contributing in mineral nutrients and activities of antioxidant
translocating photosynthates from source to the sink, enzyme34. Application of HA with SA could be an
increase net photosynthesis rate, prevent auxin effective and low-cost approach to ensure seedling
oxidation, reinforce plants’ antioxidant potential, establishment and plant growth in fields affected by
protection against oxidative stress which lead to soil drought in the early season35. Addition of humic
improve growth and drought tolerance25. Ascorbic acid to the soil raised the possibility of maintaining
acid activates maize plant´s growth and improved the growth of maize in case of lack of water
water use efficiency, also enhancing biosynthesis of available. The plant growth stimulation by humic
photosynthetic pigments and then improving yield. acids has been attributed to a hormone-like effect as
AA plays vital role in protecting plant tissues from promoting the root development and proliferation,
harmful oxidative damage by acting as reductant resulting in a more efficient water and nutrient
drought stress26. Ascorbic acid alleviates the drought absorption36.
This study aimed to investigate the changes in photosynthesis, transpiration, respiration, and the
internal tissues of the stem and leaf of maize when transport processes facilitated by the vascular
seeds and the whole plant were treated with ascorbic, bundles in the stem. Consequently, any
citric, and humic acids. The application of these enhancements or improvements in the anatomical
regulators induces changes in the anatomy of the characteristics can positively impact overall plant
plant's tissue. This change may play a crucial role in growth and development.
the vital processes occurring in the leaf, including
Materials and Methods

A field experiment was conducted over two spring materials were collected from fresh stems and leaves
seasons, in 2019 at the experimental fields of the and preserved in 70% ethanol. Cross-sections of stem
College of Agricultural Engineering Sciences, and leaf samples were prepared39 with modifications
University of Baghdad, and in 2020 at a private field according to Al-Hadeethi40.
in Babylon governorate. The relocation was due to Stem and leaf samples were preserved in 70%
COVID-19 restrictions, which prevented repetition ethanol, then cut into 4-5 cm pieces. Leaf epidermis
at the original site. A randomized complete block was prepared41, and sections were washed with
design was applied in a split-plot arrangement with distilled water and treated with a 5% sodium
three replications37. The main plots involved foliar hypochlorite solution for 5 minutes to remove
nutrition treatments with ascorbic acid (AA) and chlorophyll pigment. Samples were mounted on
citric acid (CA) at 100 mg L-1 each, as well as humic glass slides and examined under an Olympus
acid (HA) at 1 ml L-1, and a control with distilled KRÜSS light microscope, with images captured
water. Two application stages for the acids occurred using an AmScope camera. Characteristics studied
when 6 and 10 true leaves appeared. Sub-plots included the diameter of vascular bundles in the stem
included seed soaking with the same treatments, and the shapes of stomata in the leaf epidermis.
where seeds were soaked for 18 hours1,15. Data were analyzed statistically using the GenStat
Maize seeds of the Baghdad-3 cultivar were sourced program. Means were compared using the least
from the Agricultural Research Directorate, Ministry significant difference test at the probability level of
of Agriculture. Soil and crop management followed 0.05 (LSD 5%)42.
Ministry of Agriculture recommendations38. Plant
Results and Discussion

The stem cross-section of the cultivar of Zea mays Lysigenous Intercellular Space. This space expands
for the control treatment is illustrated in Fig. 1. The with the extinction of adjacent cells43,44 (Fig. 2).
cross-section of the stem consists of one layer of The leaf section consists of upper and lower
epidermis with ovoid cells followed by 1-2 layers of epidermis surrounded the mesophyll with the
hypodermal fibers in the ground tissue that consists vascular bundles45 (Fig. 3). Leaf-peeling appeared in
of ordinary parenchyma cells and many vascular the stomata, which usually consists of two guard
bundles scattered randomly. The bundle consists of cells which look thin from the center and dumbbell
the phloem and the xylem surrounded by the bundle shaped from the ends and two subsidiary cells and
sheath fibers, the xylem consists of metaxylem and short cells which is represented by cork and silica
protoxylem. The lacuna is located under the cells46,47 (Fig. 4).
protoxylem and also known as the Schizo-
Figure 1. Cross section of stem in Zea mays, DW: distilled water treatment.
Figure 2. Vascular bundle of stem in Zea mays, DW: distilled water treatment.
Figure 3. Longitudinal section of leaf in Zea mays, DW: distilled water treatment.
Figure 4. Epidermis of leaf in Zea mays, DW: distilled water treatment.
The epidermis layer consists of compact ordinary cells are observed around the bundle, especially
epidermal cells covered with cuticle and there are around the main bundles, which act as a protective
stomata. It also contains large, and thin-walled cells tissue for the bundles and support tissue for the leaf.
at which the leaf folds, known as motor cells or The lower epidermis similar to the upper
Buliform cells. The mesophyll consists of epidermis49,50.
chloranchyme cells that carry out the process of The results of the experiment for the season 2019
photosynthesis, in most plants with a single showed variations in the diameters of the vascular
cotyledon, this tissue is not distinguished into a bundles of the stem among the different treatments
palisade tissue and another spongy tissue48. compared to the control treatment. The citric acid
Vascular bundles are found in a parallel system, treatment outperformed the rest of the treatments,
usually as a result of parallel venation in monocots, reaching 186.3 µm, and in 2020 also the citric acid
in the leaf section the large bundles appear in the treatment outperformed the rest of the treatments
center and small bundles arranged laterally. The reaching 112.4 µm. Also, when soaking the seeds in
bundles are closed, with xylem located toward the acids, the citric acid outperformed, reaching 144.7
upper epidermis, forming letters Y, V, and phloem µm in 2019, and in 2020, and the humic acid
located toward the lower epidermis, sclerenchyma outperformed reaching 111.8 µm. This may be due
to the difference in the location of cultivation and the processes. However, the Krebs Cycle relies on the
quality of the seeds, as mentioned in the precise regulation of citric acid levels. Excess citric
methodology (Figs. 5 and 6). acid in the plant's water supply may disrupt this cycle
As for the interaction between the factors, in the or lead to an overabundance of phosphates,
spring season 2019, the treatment of spraying with potentially impacting cellular function and plant
citric acid and soaking with water outperformed the health.
rest of the (treatments 185.7 µm), and for the spring
The results indicated that the other treatments
season 2020, the treatment of spraying with citric
increased the diameter of the bundle compared to the
acid and soaking with humic acid exceeded, as it
control. Citric acid was the first leader in the increase
reached 147.9 µm. This may be due to the difference
among all the treatments.
in the location of cultivation and the quality of the
The treatments did not actually affect the epidermis
seeds (Fig. 5 and 6).
of the plant leaf, except for minor effects on the
The results indicate that adding citric acid to water
upper epidermis when treated with ascorbic acid and
significantly increased the diameter of vascular
citric acid, which led to a distortion in the shapes of
bundles in the plant's stem compared to the control.
some stomata compared to the control treatment.
However, it's important to note that the optimal pH
Ascorbic acid and humic acid treatment led to an
range for plant growth is between 5.5 and 7.5,
increase in the size of ordinary epidermal cells and
demonstrating the adaptability of some plants to
their walls became straight, while in the control
varying environmental conditions. Consequently,
treatment the walls were usually wavy, in addition to
excessive citric acid in a plant's water or soil can
an increase and expansion in the size of cork and
create an environment unsuitable for the plant as
silica cells (Fig. 7). The results of the study agree
shown in Figs. 5 and 6.
with the study on sorghum51, no changes were
The beneficial effects of citric acid, even in low
observed in the stem epidermis, except a few changes
doses, have been documented. A study conducted by in the size of the guard cell and diffused the prismatic
the U.S. Department of Agriculture aimed at creating crystals in the cells and around the guard cells. Also,
an insecticide derived from various plants containing the results agree with an anatomical study which
citric acid revealed that direct application of 16% showed that cultivars had a significant effect on stem
citric acid to plants had minimal impact, with anatomical traits in the diameter of vascular bundles
occasional instances of discoloration. Despite this, and xylem diameter of lupine crop52.
the plants remained healthy, suggesting that citric The effects of the solutions on the lower epidermis
acid could serve as a repellent. only appear in the treatment by ascorbic acid and
In plants, citric acid plays a crucial role in the Krebs humic acid; the treatment led to an increase in the
Cycle, where it is converted into citrate to generate size of cork and silica cells (Fig. 8).
phosphates, serving as a source of energy for cellular
Figure 5. Variations in shapes and sizes of the vascular bundles in the stem in Zea mays, DW: distilled
water treatment, AA: Ascorbic acid treatment, HA: Humic acid treatment, CA: Citric acid treatment.
Figure 6. Variations of diameter of vascular bundles in the stem of Zea mays.

Figure 7. Variations of upper epidermis in the leaf of Zea mays. DW: distilled water treatment, AA:
Ascorbic acid treatment, HA: Humic acid treatment, CA: Citric acid treatment.
Figure 8. The variations of lower epidermis in the leaf of Zea mays. DW: distilled water treatment,
AA: Ascorbic acid treatment, HA: Humic acid treatment, CA: Citric acid treatment.
Conclusion
In summary, the current study demonstrates that the generation of energy compounds, particularly ATP.
application of citric acid as a spray treatment showed Additionally, this treatment stimulated increased
notable effectiveness during both the 2019 and 2020 synthesis and accumulation of carbohydrates
maize planting seasons in the Mid-North Iraqi region throughout the plant, contributing to overall plant
in spring. The treatments examined resulted in a vigor and resilience.
significant enlargement of vascular bundles within
Moreover, the application of ascorbic acid and humic
the plant stem. This enlargement holds considerable
acid primarily impacted the lower epidermis, leading
potential for enhancing plant resilience against a
to the enlargement of cork and silica cells. This
broad spectrum of abiotic and biotic stresses.
enlargement enhances leaf strength and rigidity,
The observed enhancement in vascular bundle size enabling the plant to withstand environmental and
can be attributed to citric acid's pivotal roles in biological stresses more effectively. The
plasma membrane formation, lipid and protein composition of these cells, rich in cork and silica
breakdown, stimulation of photosynthesis, and its materials, serves as a protective shield, reinforcing
crucial involvement in the Krebs cycle for the
the plant's structural integrity and resilience to particularly under stress conditions. This approach
various stressors. could effectively enhance plant health and resilience,
ultimately leading to improved maize yields in the
Based on these findings, it is recommended to utilize
Mid-North Iraqi region during the spring planting
soak and foliar application methods for maize seeds,
season.
Acknowledgment
We are grateful to the coauthors for their of Baghdad's College of Agricultural Engineering
contributions towards finalizing the work, as well as Sciences and College of Pure Sciences for their
to the academic and technical staff of the University assistance in completing this project.
Authors’ Declaration
- Conflicts of Interest: None. - No animal studies are present in the manuscript.
- We hereby confirm that all the Figures in the - No human studies are present in the manuscript.
manuscript are ours. Furthermore, any Figures - Ethical Clearance: The project was approved by
and images that are not ours have been included the local ethical committee at the University of
with the necessary permission for re-publication, Baghdad.
which is attached to the manuscript.
Authors’ Contribution Statement
The authors confirm contribution to the paper as S. W.; draft manuscript preparation: K. J. J., H. J. H.,
follows: study conception and design: K. J. J., H. J. H. M. A., Al. M., S. W., S. M. F., B. M., R. H. Z.
H.; data collection: K. J. J., H. J. H.; analysis and reviewed the results and approved the final version
interpretation of results: H. J. H., H. M. A., Al. M., of the manuscript.
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maize (Zea mays L.) plants by improving some key 45. Crang R, Lyons-Sobaski S, Wise R. Plant anatomy: a
physico-biochemical attributes. Cereal Res Comm. concept-based approach to the structure of seed plants.
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https://doi.org/10.1556/0806.45.2017.064 46. Beck CB. An introduction to plant structure and
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‫‪Published Online First: September, 2024‬‬
‫‪https://doi.org/10.21123/bsj.2024.10020‬‬
‫‪P-ISSN: 2078-8665 - E-ISSN: 2411-7986‬‬ ‫‪Baghdad Science Journal‬‬
‫تأثير أحماض االسكوربك والستريك والهيومك في تشريح ساق وأوراق الذرة الصفراء‬
‫جزران جرد كاظم‪ ،1‬جالل حميد حمزة‪ ،2‬معزز عزيز حسن‪ ،2,3‬ميثم العامري‪ ،4‬ويليام سيرسون‪ ،5‬محمد فتحي سليمان‪ ،6‬مارتن‬
‫باتاغليا‪ ،7‬هايل زهير ريحان‪8‬‬
‫‪1‬وزارة التربية‪ ،‬تربية بابل‪ ،‬التعليم المهني‪ ،‬بابل‪ ،‬العراق‬

‫‪2‬قسم المحاصيل الحقلية‪ ،‬كلية علوم الهندسة الزراعية‪ ،‬جامعة بغداد‪ ،‬بغداد‪ ،‬العراق‪.‬‬
‫‪3‬قسم علوم الحياة‪ ،‬كلية العلوم الصرفة ‪ /‬ابن الهيثم‪ ،‬جامعة بغداد‪ ،‬بغداد‪ ،‬العراق‪.‬‬
‫‪4‬قسم علوم الحياة‪ ،‬كلية العلوم للبنات‪ ،‬جامعة بغداد‪ ،‬بغداد‪ ،‬العراق‪.‬‬
‫‪5‬جامعة والية بنسلفانيا‪ 2809 ،‬شارع سوكون فالي‪ ،‬سنتر فالي‪ ،‬بنسلفانيا ‪ ،18034‬الواليات المتحدة‪.‬‬
‫‪ 6‬قسم اإلنتاج النباتي‪ ،‬كلية علوم األغذية والزراعة‪ ،‬جامعة الملك سعود‪ ،‬الرياض‪ ،‬المملكة العربية السعودية‬
‫‪7‬قسم علوم المحاصيل والتربة البيئية‪ ،‬جامعة فرجينيا للتكنولوجيا‪ ،‬بالكسبيرغ‪ ،‬فيرجينيا ‪ ،24061‬الواليات المتحدة‪.‬‬
‫‪8‬قسم العلوم البيولوجية والبحرية‪ ،‬كلية العلوم والهندسة‪ ،‬جامعة بليموث‪ ،‬المملكة المتحدة‪.‬‬
‫الخالصة‬
‫درست التغيرات التشريحية في األنسجة الداخلية للساق والورقة عند معاملة البذور والنبات باألحماض لتعزيز النمو والتطور في الذرة‬
‫الصفراء خالل فصلي الربيع لعامي ‪ 2019‬و‪ .2020‬تم تطبيق تصميم القطاعات الكاملة المعشاة بترتيب االلوا المنشقة بثالثة مكررات‪.‬‬
‫تضمنت االلوا الرئيسة معامالت التغذية الورقية (حامض األسكوربيك (‪ )AA‬وحامض الستريك (‪ )CA‬بتركيز ‪ 100‬ملغم لتر‪،1-‬‬
‫وكذلك حامض الهيوميك (‪ )HA‬بتركيز ‪ 1‬مل لتر ‪ .1-‬تم استخدام الماء المقطر كمعاملة مقارنة‪ .‬وتضمنت االلوا الثانوية معامالت نقع‬
‫البذور بنفس المعامالت‪ .‬كشفت نتائج تحليل المقطع العرضي للساق وجود اختالفات في حجم الحزم الوعائية بين المعامالت المختلفة‬
‫بالمقارنة مع معاملة المقارنة‪ ،‬وأن التغذية الورقية باستخدام ‪ CA‬أظهرت نتائج متفوقة في كال الموسمين‪ .‬كما تفوقت البذور المنقوعة‬
‫في ‪ CA‬و ‪ HA‬على المعامالت االخرى‪ .‬كان هناك تداخل معنوي بين التغذية الورقية بالـ ‪ CA‬ونقع البذور بالماء المقطر في عام‬
‫‪ ،2019‬أما في عام ‪ 2020‬فتفوقت معاملة التداخل بين التغذية الورقية بالـ ‪ CA‬ونقع البذور بالـ ‪ HA‬على بقية المعامالت‪ .‬ومن المهم‬
‫أن نالحظ أن المعامالت كان لها تأثير محدود على بشرة أوراق النبات‪ ،‬مع مالحظة آثار طفيفة فقط‪ .‬على سبيل المثال‪ ،‬تسببت المعامالت‬
‫بالـ ‪ AA‬و ‪ CA‬في حدوث بعض التشوه في أشكال بعض الثغور في البشرة العلوية عند مقارنتها بمعاملة المقارنة‪ .‬أدت المعامالت‬
‫باستخدام الـ ‪ AA‬و ‪ HA‬إلى زيادة في حجم خاليا البشرة العادية‪ ،‬مع جدران خاليا أكثر استقامة‪ ،‬بينما في معاملة المقارنة‪ ،‬كانت جدران‬
‫الخاليا متموجة فضالً عن ذلك كان هناك زيادة وتوسع في حجم خاليا الفلين والسيليكا في النباتات المعاملة‪ .‬تسهم هذه الدراسة في فهم‬
‫التغيرات التشريحية التي تحدث في األوراق والسيقان خالل فترات اإلجهاد في موسم نمو الذرة الصفراء‪ ،‬وتسلط الضوء على التأثيرات‬
‫المحتملة للمعامالت الحامضية على وظائف النبات‪.‬‬
‫الكلمات المفتاحية‪ :‬التغذية الورقية‪ ،‬منظمات النمو‪ ،‬تشريح النبات‪ ،‬نقع البذور‪ ،‬الذرة الصفراء‪.‬‬

Effects of Ascorbic Citric and Humic Acids On Maiz

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Effects of Ascorbic Citric and Humic Acids On Maiz

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Published Online First: September, 2024

contributing to this is the selection of inappropriate tolerance by improving photosynthetic pigments,

Materials and Methods

Results and Discussion

Figure 4. Epidermis of leaf in Zea mays, DW: distilled water treatment.

Figure 6. Variations of diameter of vascular bundles in the stem of Zea mays.

‫‪1‬وزارة التربية‪ ،‬تربية بابل‪ ،‬التعليم المهني‪ ،‬بابل‪ ،‬العراق‬

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