الفهرس 
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Abstract
The present work deals with the cytogenetic effects of four stored seeds protectants against Bruchids pests (Callosobruchus maculates F and C. chinensis L.) on Vicia faba plant. The studies includes the effect of: i) dusting the seeds with different concentrations of “Malathion 1% powder” (Mal.) and fumigation of other with “Phosphine” (Phos.) for storage as synthetic insecticides active against Bruchids pest and recommended to be used in Egypt for protection of beans and grains, since1980 till now and (ii) dusting the seeds with different concentrations of either ”Lupinus termis seeds powder” (LtSP) or black cumin seeds powder (bcSP) for storage as two plant products proved to be active against the same target pest.
This study aims to evaluate the effect of the four agents after six months storage of the treated seeds on the: mitotic and meiotic apparatuses, some metric traits of the F1 &F2 plants and changes on protein profiles of M2 seeds; In addition, changes in some cytoplasmic organelles induced in the cells of Vicia faba root-tip meristems after treatment, took place by the use of TEM observations.
All experiments took place after six months of storage at room temperature under treatment with (1, 2 & 4) times the effective concentration of the four agents. These effective concentrations were:-
80 mg of Mal./100 gm of seeds.
3 mg of phos. /350 ml3 size jar filled near to the upper end and well closed.
800 mg of either LtSP or bcSP/100gm of seeds. 1- Effect on mitosis:
At the time, dusting with LtSP was without any statistically significant effect on mitotic index after treatment with all the used concentrations and those of Mal. induced highly significant reduction in only after treatment with the 4EC, both fumigation treatments with Phos. and dusting treatments with bcsp affected significant reduction in mitotic index after treatment with the EC, increased progressively to highly significant after treatment with the 2EC &4EC, which indicates that, both Phos. and bcsp were the more cytotoxic agents and LtSP was without any cytotoxic effect on cell division.
To some extent, the effect of the four agents on the phase indexes were parallel to their effect on the mitotic indexes. Dusting treatments with LtSP were without any noTable effect on the phase indexes and those of Mal. induced small increase in the metaphase index on response of the prophase index only after treatment with EC. While dusting treatment with bcSP affect the induction of progressive increase in the metaphase indexes after treatment with the 2EC &4EC on response of the prophase indexes. Treatment with Phos. were higher than those of bcSP on the phase indexes, they affected considerable progressive increase in the metaphase and anatelophes indexes on response to great progressive decrease in the prophase indexes after all concentration treatments.
The four tested materials induced highly significant and dose dependent percentages of abnormal mitoses, distributed between all the mitotic stages, mostly in metaphases followed by the ana-telophase. This means that, the four agents have mutagenic effect. Worth to mention that, dusting with ”LtSP” was the least inducer of abnormal mitoses followed by dusting with Mal. While seeds fumigated with Phos. gas was the most destructive i.e. the most mutagenic agent and LtSP was the least.Treatment with each of the four tested agents induced different types of abnormality, can be arranged according to their abundance into cells with chromosomes stickiness > disturbance > lagging > fragments and/or breakage followed by cells with micronuclei and irregular prophases.
Dividing cells examination of the root-tip meristems revealed that, dusting treatment with LtSP followed by Mal. were the least inducer of chromosome stickiness which means that, they are the least chromotoxic agents compared with Phos. or bcSP.
Also, dividing cells examination of the root-tip meristems clears that, fumigation with Phos. Induced the higher percentage of disturbancy which means that it has the more toxic effect on the spindle i.e. it have the more turbagenic effects. Clumped metaphases as a type of disturbance were recorded only after treatment with the two synthetic pesticides while splitting of the spindle was observed only after treatment with the two plant products.
Relative to the scored number (metaphase +anaphase) cells, dusting treatment with each of the three dusters in their effective concentration didn’t show any significant clastogenic effect, while fumigation treatment with Phos. affected significant percentage after the same concentration (EC). Higher concentrations treatment of the four agents induced significant to highly significant percentages of (Metaphase + anaphase) cells suffering from structure aberrations, which means that higher concentrations are the more clastagenic mode of action. Generally, dusting treatment with LtSp followed by the treatment with Mal. in the lower used concentration were the least clastogenic agent while dusting with bcSP in its higher concentration (4EC) was the most destructive agent Interphase cell with micronuclei was the only type of abnormality observed in the root tip meristems after treatment with the four agents. Slight, dose dependent were their percentages. Both Mal. and LtSP affected significance only after treatment with the higher used concentration. Higher effects were observed after treatment with the others (Phos. and bcSP).
2- Effect on meiosis:
The four tested agents induced highly significant percentages of abnormal PMCs/ plant increasing dose dependent were their percentages after treatment with the two lower concentrations of Mal. and after treatment with the three concentrations of the others. Dusting treatment with the 4EC of Mal. was the least inducer of the abnormal PMCs/ plant.
The analyzed data revealed that, the percentages of abnormal PMCs in the first Meiotic division were higher than those in the second meiotic division, unless those of the EC treatment with either Phos. or bcSP.
The four agents’ treatment induced wide types of abnormal PMCs covering all stages of the meiotic division. These types were similar to those recorded after mitosis in addition of multi polarity.
Data revealed that, all concentration fumigation treatment with Phos. affected the higher percentages of PMCs. suffering from stickiness of the chromosomes and PMCs. with chromosome kinetic abnormalities and treatment with the EC of LtSP affected the least percentages of both.
Statistical analysis of the percentages of (metaphase I & II + anaphase I & II) PMCs/ to the scored number revealed that, Mal. was the least inducer of PMCs with structural aberrations, it affected significance only after treatment with the higher used concentration, followed by LtSP which affected significance after treatment with the two higher concentrations while Phos. followed by bcSP were of the most destructive agents, they affected significance after treatment with all concentrations.
Slight percentages of abnormal tetrads were observed after some concentration treatments with the four agents. The higher percentages were observed after treatment with Phos. followed by bcSP. Pentads and unequal tetrads were the types of abnormality observed. Pentads were observed after treatment with some concentrations of either Mal., Phos or LtSP and unequal tetrads after treatment with the higher used concentration of bcSP.
Slight concentration dependent percentages of abnormal pollen grains were observed after treatment with the four agents. LtSP resembled Mal., were the least effective, they affected significance only after treatment with the 4EC. Higher effects were observed after treatment either with Phos. or bcSP, they affected significance after treatment with 2EC, increased to highly significant after treatment with the 4EC. Small pollen grains and deformed pollen grains, were the types of abnormality observed.
3- Effect on some metric traits:
While, dusting treatments with either Mal. or LtSP were without any significant effect on the percentage of plant survival/pot, those of dusting treatment with bcSP similar to fumigation treatment with Phos. affected significance only after treatment with the 4EC.
Dusting treatment with LtSP resembled that with Mal. they affected highly significant decrease of 50 days old plants height of the F1 generation only after treatment with the 4EC, lowered to significant after 100 days from sowing and 50 & 100 days from sowing of the F2 generation plants. Higher effects were observed after treatment either with Phos. or bcSP. Phos. induced highly significant reduction in plants height of 50 & 100 days old plants of the F1 generation only after treatment with 4EC, increased to significant and highly significant reduction in plants height of the 50 & 100 days old plants of the F2 generation after treatment with 2EC and 4EC. Those of bcSP affected highly significant reduction in plants height of 50 days old plants of the F1 generation and 50 &100 days old plants of the F2 generation plants after treatment with 4EC.
Dusting treatment either with Mal. or LtSP induced significant decrease in the number of pods and seeds/ plant and weight of seeds/plant, after treatment with the 4EC for both F1 & F2 generations. While fumigation treatment with Phos., induced significant to highly significant decrease in this parameters, after treatment with the two higher concentrations (2EC & 4EC) of the F1 & F2 generation plants. Higher effects were observed after treatment with bcSP, since it affected highly significant, dose pendent decrease in the number of pods and seeds/plant of the F1 & F2 generation plants and weight of seeds/plant of the F1 generation plants. But, those of weight of 100 seeds/plant affected significance after treatment with only the higher used concentration (4EC) either of Phos. or bcSP.
All the above metric traits data indicates that, both Phos. and bcSP were the more inducers of genetic variability and LtSP and Mal. Were the least.
4- Effect on M2 seed storage protein banding patterns:
The four tested agents induced many qualitative variations in the M2 seed storage protein banding patterns compared with the control. These variations include: alterations in bands relative mobility;appearance of some bands; disappearance of other band and subfractionation of some bands at the lower molecular weight.
The different treatments with either Phos. or bcSP affected the higher number of disappearance of bands and those of Mal. and LtSP affected the least.
Treatment with the different concentrations of either phos. or bcSP induced the higher number of newly elaborated bands and those of LtSP induced the least.
On this account, the revealed variations in this study showed that, both Phos. and bcSP are the more potential mutagenic agents and LtSP followed by Mal. are the least.
5- Effect on the cytoplasmic organelles:
Transmission electron microscope investigation of some cytoplasmic organelles such as mitochondria, endoplasmic reticulum and dictyosomes in addition to vacuoles in root-tip meristems show that the four tested agents affected the concerned organelles in almost similar way but with variable extents.
Some mitochondria were ruptured, swollen, elongated or even with lysised content. While some other mitochondria could keep its normal form even after the treatment with the higher concentration of each of the four tested agents.
Dictyosomes were easily detected after dusting with Mal. and fumigation with bcSP. Some dictyosomes were distorted or with swollen sacs as detected after fumigation with phos. and EC bcSP.
Lysosomes as modified form of Golgi bodies were easily detected after fumigation with 2EC of phos. and after dusting with the same concentration of bcSP. Presence of lysosomes gives an alarm of the damaging affect of the tested agents.Treatment with each of the four agents affected endoplasmic reticulum in many ways. Sometimes it was wrapped around organelles or ruptured or even dilated and can hardly be detected.
Vacuoles mostly autophagic were detected in different sizes and numbers after treatment with each of the four agents under investigation.
Hard was the effect of treatment with the 4EC of each of the four agents, but even, many organelles in the cytoplasm can keep their normal form and can be easily distinguishable.
The occurrence of a large autophagic vacuoles after treatment with 2EC of bcSP and large number of vacuoles of different sizes after those of Phos., in addition to the induction of lysosomes and distorted dictyosomes after treatment with the same concentration of either Phos. or bcSP indicates that, they were the more effective on the structure and function of the cytoplasmic organelles and those of Mal. and LtSP were the least.