RU2180474C1 - Method for presowing treatment of crop seeds for synchronizing cell division in root meristem - Google Patents

Abstract

FIELD: agriculture and plant cultivation; selective and genetic studies for producing valuable mutants of farming plant. SUBSTANCE: physical method used for acting on biological objects such as seeds involves their exposure to shock waves in water medium at temperature of 2 C and production of pulses of 155-554 N•cm^-20 followed by dipping seeds in water at given temperature for 12 h. EFFECT: enhanced efficiency of cell division synchronization; elongated phases of mitotic cycle required for studies. 4 dwg, 2 tbl, 1 ex

Description

 The invention relates to agriculture and crop production, in particular to physical methods of influencing biological objects, in particular seeds, and can be used in selection and genetic work aimed, for example, to obtain valuable mutants of agricultural plants.

 Cells at different phases of the mitotic cycle exhibit unequal sensitivity to mutagens. By synchronizing the meristem cells to the sensitive phases of the cell cycle and changing the duration of the phases of mitosis, it is possible to increase the sensitivity of cells to mutagens, i.e. contribute to an increase in the frequency and spectrum of induced mutations, which is important for selection and genetic work.

 A known method of processing seeds of agricultural crops, in which the seeds are placed in water and create a shock wave with a front pressure of 5 ... 40 MPa due to detonation of the explosive [1]. The magnitude of the pressure and its duration are regulated by the mass of the explosive and the distance between it and the surface of the seeds. The application of the proposed method allows the processing of seeds under the condition of volumetric compression for a short period of time without destroying the seeds as biological objects. The specified method leads to increased crop yields. This effect has a stimulating effect on the growth of the plant, however, the resting state in the meristem after the shock-wave treatment is short-lived.

A well-known physical impact method for synchronizing cell divisions in the germinal meristem of cereals is also a method of treating seeds with an electromagnetic field with a frequency of 59.0-62.6 GHz at a power flux density of 100-300 μW • cm ^-2 in a continuous mode for 10-30 minutes [2]. The disadvantages of this method are the relatively low efficiency of synchronization of divisions (no more than 1.5 ... 2.0 times) and the impossibility of affecting individual phases of the mitotic cycle.

There are also known methods for synchronizing mitoses in the barley meristem, consisting in the action of 5-aminouracil (5-AC) [3], or 5-AC and colchicine [4] on seedlings or soaked seeds, followed by exposure of objects in water at 2 ^o С for 12 hours. These substances can cause long-term effects in the genetic apparatus of plants. These methods are laborious, multi-stage, long. They require the use of expensive reagents and lead to injury when sowing seedlings in a state of active germination. However, the synchronization effect with this processing method is less than in the previous one.

 The aim of the invention is to increase the effect of synchronization of cell divisions and prolongation of individual phases of the mitotic cycle for conducting genetic selection work.

This goal is achieved by the fact that dry seeds are treated with a shock wave in an aqueous medium at 2 ^o With the creation of pulses 155 ... 554 H • s • m ^-2 followed by their soaking in water at a given temperature for 12 hours. The momentum is controlled by the mass of the explosive and the distance between it and the surface of the seeds. The aftereffect of treatment lasts for 30 days.

A comparison of the known solutions with the claimed one showed that it is characterized by keeping the seeds at 2 ^° C for 12 hours after the shock wave treatment in order to enhance the stress response and the dormancy state in order to increase the efficiency of synchronization of cell divisions in the meristems and to prolong individual phases of the mitotic cycle. Carrying out shock wave treatment in water at 2 ^o C is necessary to ensure a stressful situation at the time of passage of the shock wave. Short soaking during shock wave treatment allows drying of seeds and their subsequent storage without changing germination. A long period of aftereffect is a significant advantage of the proposed method, since it increases the time for choosing the optimal timing of sowing and (or) the subsequent exposure of the mutagen to the seeds.

Example. Dry seeds of agricultural crops (buckwheat) were subjected to shock wave treatment in water at 2 ^o C. The value of the generated pulse was changed in the range from 100 ... 800 N • s • m ^-2 . The specific impulse value (J, N • s • m ^-2 ) was calculated by the formula [5] J = P • Θ, where P is the pressure at the front of the shock wave, MPa; Θ the characteristic time that determines the intensity of the pressure drop at a given point over time.

,
где Q - масса заряда взрывчатого вещества, кг;
R - расстояние от центра взрыва до поверхности семян, м.

,
where Q is the mass of the explosive charge, kg;
R is the distance from the center of the explosion to the surface of the seeds, m

После ударно-волновой обработки семена выдерживали в воде при 2^oС в течение 12 ч, что соответствует оптимальным режимам, приводимым в работах [3, 4]. В дальнейшем семена высушивали на воздухе при комнатной температуре до тех пор, пока их масса не станет постоянной.

After the shock wave treatment, the seeds were kept in water at 2 ^° С for 12 h, which corresponds to the optimal conditions given in [3, 4]. Subsequently, the seeds were dried in air at room temperature until their mass became constant.

After drying, mature, matured fruits were selected and germinated at 25 ^{° C.} in a humid atmosphere. The roots of seedlings, starting from the age of 48 h, were fixed in acetic acid with ethanol (1: 4) for 12 h with an interval of 2 h. The material was stained in aceto-oresin. Squeezed preparations were examined at 600-fold (vol. 40 ^x , approx. 15 ^x ) magnification and mitotic and phase indices (MI, PH) of apical root meristems were determined. Since significant changes in the average MI were detected, to determine the synchronism of mitoses, a relative increase in the maximum MI was used compared with the average in each experiment and the results obtained in the experimental plants were compared with the control.

The change in MI of buckwheat seedlings in dynamics is given in table. 1, where data for pulses equal to 155 ... 554 H • s • m ^-2 are reflected. Pulses from 100 to 155 N • s • m ^-2 did not affect cell division, and MI did not differ from the control results. Impulses above 554 N • s • m ^-2 were fatal to seed embryos.

, а среднее -

. Относительное увеличение максимального МИ равнялось 1,20. При J=155 Н•с•м^-2 среднее значение МИ составило

. Максимальное значение МИ в 52 ч составляло

. Его относительное увеличение по сравнению со средним в данном варианте опыта составило 1,77, то есть синхронность делений превысила контроль на 47,5%. При J=401 Н•с•м^-2 среднее значение МИ составляло

. Максимальное значение МИ возросло до

через 50 ч. Относительное увеличение МИ соответствовало 3,30, что указывало на увеличение синхронности клеточных делений в 2,8 раза по сравнению с контролем. При воздействии J=554 Н•с•м^-2 среднее значение МИ уменьшилось до

, что было на 31% ниже контроля. Максимальное значение МИ соответствовало 88,1%, его относительное увеличение составило 1,57. Синхронность митозов возросла на 30,8% относительно контроля.The results are shown in table. 1 show that the MI fluctuations of the control buckwheat seedlings were insignificant. The maximum value was reached at 50 h and amounted to

and average

. The relative increase in maximum MI was 1.20. At J = 155 N • s • m ^{-2 the} average MI value was

. The maximum MI value at 52 hours was

. Its relative increase compared with the average in this experiment was 1.77, that is, the synchronization of divisions exceeded the control by 47.5%. At J = 401 N • s • m ^{-2 the} average MI value was

. The maximum value of MI increased to

after 50 h. A relative increase in MI corresponded to 3.30, which indicated a 2.8-fold increase in the synchronism of cell divisions compared to the control. When exposed to J = 554 N • s • m ^{-2, the} average MI decreased to

, which was 31% below control. The maximum MI value corresponded to 88.1%, its relative increase was 1.57. The mitosis synchronism increased by 30.8% relative to the control.

клеток находились на стадии профазы, а у 52-56-часовых проростков начали преобладать клетки на стадии мета- и анафазы. Их количество превышало

от общего числа клеток. Количество телофазных клеток начинало увеличиваться у 54-56-часовых проростков и достигло максимума у 58-часовых проростков. Максимум МИ соответствовал 50-52-часовым проросткам, когда или преобладали профазные клетки (

), или количество клеток в стадии про-, мета-, анафазы было приблизительно одинаково (соответственно 257, 285,

), а в стадии анафазы - несколько ниже,

(фиг. 1,а).Marked fluctuations in phase indices (Fig. 1, a). In 48-50-hour control seedlings more

cells were at the prophase stage, and in 52-56-hour-old seedlings, cells at the meta- and anaphase stages began to predominate. Their number exceeded

of the total number of cells. The number of telophase cells began to increase in 54-56-hour-old seedlings and reached a maximum in 58-hour-old seedlings. The maximum MI corresponded to 50-52-hour seedlings when prophase cells prevailed (

), or the number of cells in the pro-, meta-, anaphase stage was approximately the same (respectively 257, 285,

), and in the anaphase stage - somewhat lower,

(Fig. 1, a).

) у 54-часовых проростков, указывающее на задержку большинства клеток на данной стадии. Эти нарушения были временными, так как уже через 56 часов отмечался максимум телофазных клеток (

). Максимум МИ соответствовал прохождению метафазы и особенно анафазы большинством клеток, что также указывало на изменение в смене фаз митоза.After treatment at J = 155 N • s • m ^{-2, the} amplitude of the FI oscillations (Fig. 1, b) was more significant than in the control. A significant increase in the number of anaphase cells (up to

) in 54-hour seedlings, indicating a delay of most cells at this stage. These disorders were temporary, since already after 56 hours there was a maximum of telophase cells (

) The maximum MI corresponded to the passage of metaphase and especially anaphase by most cells, which also indicated a change in the phase change of mitosis.

, 50 часов -

) и анафазы (52 часа -

, 54 часа -

). Первые телофазы появились только через 52 часа, тогда как в контроле они присутствовали постоянно.Processing J = 401 N • s • m ^-2 contributed to the most significant changes in FI (Fig. 1, c). The number of cells at the prophase stage increased (48 hours -

50 hours -

) and anaphase (52 hours -

54 hours

) The first telophases appeared only after 52 hours, while in the control they were constantly present.

через 52 и 54 часа и анафазных клеток до

через 54 часа. Количество профазных клеток 48 часов в опыте соответствовало контролю (

и

соответственно). Первые телофазы появились только через 54 часа, а через 56 часов составляли уже

.Under the action of J = 554 N • s • m ^{-2, the} amplitude of the FI oscillations also exceeded the control (Fig. 1, d). Characteristic increase in the number of metaphase cells to

after 52 and 54 hours and anaphase cells to

after 54 hours. The number of prophase cells 48 hours in the experiment corresponded to the control (

and

respectively). The first telophases appeared only after 54 hours, and after 56 hours they were already

.

 When analyzing anomalies of divisions by the anaphase-telophase method in all variants of the experiment, chromosome aberrations (bridges and fragments) were not found. Thus, pre-treatment affects the passage of the mitotic cycle, but does not cause damage to the chromosomes, that is, it itself does not cause a mutagenic effect and can be used to synchronize cell divisions.

 In special experiments, the aftereffect of the treatment was investigated. Seeds were processed according to the described method, 3 batches of seeds were stored for 1, 30 and 40 days, germinated and determined MI at the age of 48-58 hours. The data are given in table. 2. It is seen that the aftereffect of the treatment is stored during storage of seeds up to 30 days.

 As follows from the table. 2, when storing seeds for 1 day after treatment, the maximum synchronization effect is detected (1.47 ... 2.75 times). When stored for 30 days, the effectiveness of the effect remains virtually unchanged. When stored for 40 days, the effect of mitosis synchronization is reduced and amounts to 9-98%.

Sources of information
1. RF patent 2083073 A method for pre-sowing seed treatment of agricultural crops. Atroshchenko E.S., Khryanin V.N. et al.

 2. A. S. 1692408 "Method for pre-sowing seed treatment for synchronization of cell divisions in the germinal meristem of grain crops. N.G. Shestopalova, L.G. Golovina and others.

 3. Bendroraitite L.P. Cytology and Genetics, 1978, v. 12, 3 - S.195-199.

 4. Artemyeva V.V. Abstract of dissertation for the competition can degree biol. sciences. L., 1987, 22 p.

 5. Pikhtovnikov R.V., Zavyalova V.I. Stamping sheet metal by explosion. M.: Mechanical Engineering, 1964. - S. 42-43.

Claims (1)

A method of presowing treatment of seeds of agricultural crops to synchronize cell divisions in the root meristem, including a single exposure to seeds by a shock wave in an aqueous medium, characterized in that the shock treatment of seeds is carried out in water at 2 ^{° C.} with the creation of pulses 155.. . 554 N • s • m ^-2 followed by soaking the seeds in water at this temperature for 12 hours

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