RU2798519C1 - Method for accelerated production of healthy virus-free plants of berry crops in vitro - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: use of the combined spectral range of LED lighting to accelerate the processes of increasing biomass and increasing the multiplication factor and, accordingly, obtaining more microplants from one parent sample by improving the quality of artificial lighting when growing plants, as well as the concentration of selected phytohormones, for the stage of micropropagation and of rhizogenesis, the developed modes of sterilization of plant explants when introducing berry crops into in vitro conditions, the inclusion of real-time RT-PCR molecular diagnostic methods to accurately confirm the absence of viruses, and the selection of virus-free mini-plants before mass micropropagation of berry plants cultures under in vitro conditions.

EFFECT: enhancement of the production.

1 cl

Description

The invention relates to the field of biotechnology, crop production, agriculture and can be used in accelerated reproduction and obtaining promising plant genotypes in in vitro culture, responsive to artificial LED lighting using agrobiotechnosystems, in agrobiophotonics and in technologies for obtaining a healthy virus-free planting material.

Known universal modified nutrient medium M-S for cloning strawberry microplants varieties Irma, Elizabeth in vitro, which is a way to create a universal optimized nutrient medium, in which the Murashige-Skoog nutrient medium reduces the concentration of 6-BAP from 0.5 ml/l to 0 35 ml/l, followed by stimulation of the production of cytokinins by plants, the content of indoleacetic acid (IAA) 0.2 mg/l while reducing the concentration of the carbohydrate component (sucrose) to 25 g/l. The proposed universal modified nutrient medium for in vitro use at the stage of strawberry multiplication (cultivars Irma, Elizaveta) made it possible to increase the multiplication factor by 34.05 (RU 2747781, IPC A01H 1/00).

The disadvantage of this method is the inability to use it for other plant species, such as blackberries or raspberries, since it is known that each plant requires its own individually selected specific compositions of nutrient media and growth regulators for growing regenerative plants. And also in the technology it is impossible to get planting material that is healthy and tested for the absence of viruses.

A known method of growing strawberry plants using the in vitro method is that in rooted microplants in the period from the beginning of August to February, with a frequency of 1-1.5 months, the roots and leaves that have formed and begin to die are completely removed, and they are in the form of vegetative kidneys are planted repeatedly and differentially on nutrient media containing auxin, taking into account the diameter of the kidney. This allows you to completely eliminate the loss of material in a period unfavorable for adaptation and significantly equalize the parameters of plant development by the time of planting in non-sterile conditions. The planting of the material accumulated in this way is carried out starting from April in a greenhouse using upper fine irrigation or temporary film cover to maintain high humidity. As a substrate, pre-moistened peat or a peat-sand mixture without any temperature treatment is used (RU 2762979, IPC A01H 4/00)

A known method of intensifying the germination of radish seeds under pulsed lighting, which turns on flashlight. On the 7th day of germination in the dark of radish seeds, seedlings are fed with a mineral solution of the composition (mg/l): N-NH₄- 0.5; P - 4.1; K - 27.5; Ca - 10.0; Mg - 2.4; S - 3.0; Fe - 0.094; Mn - 0.014; B - 0.016; Cu - 0.003; Zn - 0.013; Mo - 0.003. Next, they are irradiated with LED lamps at an intensity of generated photons of 140 µmol/m²s with a pulse duration of 1 s of illumination and 3 s of a pause of darkness, with the characteristics of a polychrome spectrum: ultraviolet 380 nm - 1.5%, blue 440 nm - 23.8%, green 520-530 nm - 6%, red 640 nm - 61 .5%, far red 740 nm - 7.2% from the 7th to the 14th day of round-the-clock illumination of seedlings until microgreens are obtained (RU 2735868, IPC A01C01/00; A01G07/04; A01C 21/00)

The disadvantage of this method is the use of the developed composition of the spectral range only to stimulate the germination of radish seeds, and not to actively increase biomass and photosynthesis, and as you know, each plant and at each phase of phenological development requires its own individually selected spectral growing conditions.

In the article L. A. Gud, E. A. Kalashnikova, I. G. Tarakanov (Internet link: http://dx.doi.org/10.24419/LHI.2304-3083.2019.2.09 Gud, L. A., E A. Kalashnikova, I. G. Tarakanov Influence of light of different spectral range on blackberry and raspberry morphogenesis in vitro // Lesokhoz.inform.: electronic network journal - 2019. - No. 2. - P. 97-102. URL: http://lhi.vniilm.ru/), the authors studied the effect of light of different spectral ranges on the morphogenesis of blackberries and raspberries in vitro. It has been established that microshoots of raspberry and blackberry, in order to increase the growth of shoots, should be cultivated under in vitro conditions under blue spectrum LED lamps (CD-S) or a white chip with a phosphor (CD-CHLB).

But these lighting conditions have a positive effect on the multiplication factor, but only in blackberries. The disadvantage is also that the cultivation of plants was studied only on separate spectra, and not combined on one shelf, which accelerates the growth and development of plants, with the correct selection of a combination of spectra.

Therefore, there is a need to develop an accelerated method for obtaining a healthy virus-free planting material for berry crops (blackberries, raspberries) under in vitro laboratory conditions using molecular diagnostic methods for the absence of viruses and modern methods of agrobiophotonics using combined LED lighting spectra, which make it possible to increase the multiplication factor of plants at micropropagation of plants.

The task facing the author is to develop a method for the accelerated production of healthy planting material for berry crops using biotechnological methods of micropropagation, molecular genetic diagnostics (PCR testing or using polymerase chain reaction), molecular diagnostics (virus free) and methods of agrobiophotonics.

The problem is solved by the proposed method for obtaining healthy plants based on the use of methods of plant cell biotechnologies, molecular genetic methods for diagnosing plant material for the absence of viruses and the use of agrobiophotonics methods to increase the growth rate and development of mini-plants in the process of microclonal propagation and cultivation in laboratory conditions in vitro.

The essence of the proposed method is the use of the combined spectral range of LED lighting to accelerate the processes of increasing biomass and increasing the multiplication factor and, accordingly, obtaining more microplants from one parent sample by improving the quality of artificial lighting when growing plants, as well as the concentration of selected phytohormones for the stage of microclonal propagation and rhizogenesis, the developed modes of sterilization of plant explants when introducing berry crops into in vitro conditions, the inclusion of real-time RT-PCR molecular diagnostic methods in the technology to accurately confirm the absence of viruses, and the selection of virus-free (virus free) mini-plants before mass microclonal propagation of plants berry crops under in vitro conditions.

Implementation of the method

As plant explants, buds with plant apical meristems are used, which are disinfected by stepwise sterilization. To do this, plant explants are initially treated in a soap solution, then washed several times with distilled water, placed in a solution of foundationazole for 30-60 minutes, then in a laminar box they are placed in 70% alcohol for 1 minute, and then in the main sterilizing solution, which is either whiteness 100% when exposed for 5 minutes or sublimate 0.1% when exposed for no more than 3-5 minutes or lysoformin 3000 5% for 5 minutes. Next, the plant explants are washed with autoclaved distilled water three times for 15 minutes and the meristems are isolated using a stereomicroscope under sterile conditions in a laminar box, then the meristems are placed on a modified Murashige and Skoog nutrient medium, using phytohormones to activate plant growth from the meristem with the addition of 6- BAP (6-benzylaminopurine) in an amount of 0.5 to 1 mg/l, cultivated in a light room at a temperature of 24°C with a photoperiod of 16 hours day 8 hours night. Plants obtained under sterile conditions are further tested by molecular diagnostic methods for the absence of viruses and pure plants that are free of viruses are selected only after it has been proven that the plants are healthy and virus-free and are used for subsequent mass micropropagation with additional selective testing of some plants after microcloning. For this, reverse transcription PCR analysis is used, this analysis is carried out using a real-time PCR system (real-time). To do this, RNA is initially isolated from plant material using an isolation kit, according to the attached protocol: from the plants obtained in in vitro culture, the lower leaves are cut off under sterile conditions, weighing about 50-70 mg and ground in a mortar with a pestle along with 400 μl of lysing solution. Next, the resulting homogenate is transferred into pre-labeled plastic tubes (one sample per tube) and thermostated at a temperature of 65°C for 20 minutes, then centrifuged for 10 minutes at 2000 rpm and the resulting supernatant is transferred into a clean plastic tube. In the same tube, add the precipitation reagent in an equal volume and mix on a vortex, centrifuge the tube again for 15 minutes at 13,000 rpm. With a separate tip for each sample tube, the supernatant is removed, and 500 µl of washing buffer No. 1 is added to the precipitate and shaken on a vortex. The tubes are centrifuged for 5 minutes at 13000 rpm, the supernatant is again removed according to the same principle and 300 µl of wash buffer No. 2 is added to each tube, shaken on a vortex. And then the tubes are centrifuged for 5 minutes at 13,000 rpm, the supernatant is removed, and the resulting precipitate is dried by opening the tube lid for 5 minutes at a temperature of 65°C. To the dried sediment add 50 μl of buffer to dissolve the isolated RNA, shake the tubes on a vortex for 5 seconds, then thermostat for 5 minutes at a temperature of 65°C and shake the tubes on a vortex for 5 seconds. The RNA isolated from the leaf samples is ready for further work on reverse transcription, which included the preparation of a preliminary general mixture. To do this, a mixture for reverse transcription is prepared in a separate tube: 1 µl of the sum of primers included in the kit and 0.5 µl of reverse transcriptase are added to 2 µl of RT-buffer, thoroughly mixed on a vortex. All further work is carried out in a clean area. Clean plastic tubes are labeled for each sample and 3.5 µl of the mixture is added to each for reverse transcription, then an RNA sample is added to each and the mixture is shaken in the tube on a vortex for 5 seconds, droplets are precipitated by short-term centrifugation. The tubes are incubated in an incubator at 40°C for 40 minutes, and then reverse transcriptase is inactivated by heating at 95°C for 10 minutes. Add 80 µl of cDNA dilution buffer to all tubes, mix the tubes on a vortex, and precipitate drops from the walls of the tubes by short-term centrifugation. The cDNA preparation is ready and then it is used for PCR amplification. Alternatively, the cDNA preparation is placed in a -20°C freezer and used later.

Next, amplification is carried out. Mark on the side of the test tubes included in the diagnostic kit without touching the paraffin layer, and add 10 µl of Taq polymerase and then 20 µl of mineral oil to each. Further work is carried out in a sterile clean area. Add to the test tubes, without touching the paraffin layer, 5 µl of DNA from previously isolated plant samples, except for negative and positive control tubes. 5 µl of the negative control sample that has passed the DNA isolation step is added to the negative control tube, the same amount of the positive control sample included in the kit is added to the positive control tube. The tubes are centrifuged at 1000 rpm for 3-5 s to precipitate all components of the mixture to the bottom of the tube. Next, the tubes are placed in the amplifier for real-time PCR reaction.

Based on the results of RT-PCR diagnostics, pure virus-free plants are selected and microclonally propagated to ensure the production of mass-quality elite planting material. Mini plants are placed on Murashige and Skoog modified nutrient medium for micropropagation with the addition of phytohormones 0.5-1 mg/l 6-benzylaminopurine (6-BAP) and 0.1 mg/l IBA (indolyl-butyric acid) to induce the formation microplants. After 30-45 days, from 3 to 30 new microplants are obtained from one cultivated mini-plant.

To achieve such a high multiplication factor, cultivation is carried out on special racks with an adjustable combined spectral range of LED lighting, which has a programmable controller that allows you to adjust the spectral range on each shelf and simultaneously use all the spectra on one shelf, adjusting and selecting a different combination of blue on each shelf at the same time. , red and white (green) in the spectral composition for the growth and development of plants. This has huge advantages for improving the technology of accelerated micropropagation in comparison with the use of fluorescent lamps or the use of phytolamps of only one spectrum, without the possibility of regulation.

Cultivation of mini-plants was carried out on a rack with a combined spectral range of LED lighting according to the ratio on one shelf in the spectrum of red, white and blue: 79%, 1%, 20%. Fluorescent lamps served as control in the experiment. When grown in this combined spectral range, raspberry and blackberry plants were maximal in terms of biomass development and multiplication factor.

Next, the obtained conglomerates of microplants were separated in a laminar box and passaged on new modified Murashige and Skoog nutrient media to induce rhizogenesis or root formation with the addition of 1 mg/l of indolyl-butyric acid (IMA) or 1 mg/l of indolyl-acetic acid (IAA). After 6-8 weeks of cultivation, plants with a well-developed root system and above-ground biomass were transplanted into the soil substrate and grown under stable conditions: temperature 20-22ºС, relative humidity 70-80%, photoperiod 16 h. pots with a volume of 0.25 l with a mixture of peat (pH 5.0-5.6) and perlite in a ratio of 2:1. Mini-plants were carefully removed from the culture jar using tweezers, without damaging the plant and root system. The regenerated plant was cleaned of the nutrient medium and transferred with tweezers to the soil recess in the pot, while the soil mixture was moistened in advance. After that, the roots were lightly covered with soil, but the root collar of the regenerated plant was left at the level of the soil mixture in the pot. The planted plants were covered with a transparent cap made of plexiglass, a container with water was placed inside to maintain humidity and better plant survival. Cultivation was carried out for 14 days. Next, the cap was gradually opened at first for a few minutes, the next days - for several hours, and then the plants were grown without a cap for another 14 days.

Thus, virus free berry crops, free from viruses and diseases, will become more efficient and cost-effective planting material. An additional positive advantage of this development is the ability to save energy through the use of LED racks.

Claims (1)

A method for obtaining healthy virus-free plants of berry crops in vitro, which consists in using buds with plant apical meristems as plant explants, which are disinfected by stepwise sterilization, for this, plant explants are initially treated in a soapy solution, then washed with distilled water, placed in a sterilizing solution , then the plant explants are washed with autoclaved distilled water and the meristems are isolated using a stereomicroscope under sterile conditions in a laminar box, then the meristems are placed on a modified Murashige and Skoog nutrient medium, using phytohormones to activate plant growth from the meristem with the addition of 6-BAP and cultivated in light plants obtained under sterile conditions are tested using molecular genetic diagnostic methods for the absence of viruses and pure virus free plants are selected and used for subsequent mass micropropagation, while diagnosis for the absence of viruses is carried out using real-time PCR analysis, for this, leaves are cut off from plants obtained in in vitro culture under sterile conditions and triturated with a lysing solution into test tubes and thermostated, then centrifuged, and the resulting supernatant is transferred to a test tube, where the precipitation reagent is added in an equal volume and mixed, centrifuged again and removed the supernatant is added to the sediment, the washing buffer is added and shaken on a vortex, then it is centrifuged and the supernatant is removed again according to the same principle and the washing buffer is added again, the tubes are shaken and then centrifuged, the supernatant is removed, and the resulting precipitate is dried by opening the lid of the tube, a buffer is added to the dried precipitate to dissolve the isolated RNA, then shaken, then thermostated and shaken again, the RNA isolated from leaf samples then goes through the reverse transcription stage, for this a reverse transcription mixture is prepared, including an OT buffer, primers, and reverse transcriptase, then mix, after which the reverse transcription mixture is added to each empty tube, and the RNA sample is added and the mixture is shaken, after which it is incubated, and then the reverse transcriptase is inactivated by heating, while dilution buffer is added to all tubes and the contents of the tube are mixed, then amplification is carried out, for this, without touching the paraffin layer, Taq polymerase and mineral oil and DNA from previously isolated plant samples are added to each tube, the tubes are centrifuged and placed in an amplifier for real-time PCR reaction, the results are recorded automatically using software According to the results of RT-PCR diagnostics, pure virus-free plants are selected and microclonally propagated, for this, mini-plants are placed on a modified nutrient medium of Murashige and Skoog with the addition of phytohormones 6-benzylaminopurine and indolyl-butyric acid to induce the formation of microplants, which are cultivated on a rack with a combined spectral range of LED lighting, including red, white and blue spectra on the same shelf, then the resulting conglomerates of microplants are separated in a laminar box and pass them on new modified nutrient media Murashige and Skoog to induce rhizogenesis with by adding indolyl-butyric acid or indolyl-acetic acid, after that, plants with a well-developed root system and above-ground biomass are transplanted into a soil substrate and grown.