The effect of growth regulators on the formation of Solanum tuberosum L. micro-plants in vitro

doi:10.21203/rs.3.rs-2859120/v1

Micro-plants of different varieties of potatoes (Solanum tuberosum L.) of different ripening times: very early ‘VB Venta’, early ‘Goda’, medium ‘VB Meda’ and very late ‘VB Aista’, as well as growth regulators IAR (indole-3-acetic acid) and kinetin (6-furfuryl aminopurine). The length (cm) and weight (g) of the potato micro-plants were evaluated during testing. Microplant explants were grown in modified MS nutrient medium with different amounts of growth regulators. The results of auxin (IAR) studies showed that the highest number of micro-plants grew from the ‘VB Meda’ variety (10.03 ± 0.96 cm), and the lowest number of micro-plants grew from ‘VB Venta’ (6.2 ± 0.75 cm); the explosives of the ‘VB Meda’ variety (0.218 ± 0.09 g) formed the largest mass of micro-plants. At a concentration of 1.75 ml l^− 1 auxin, a sudden increase in the length and weight of the micro-plants was observed. The results of experiments using kinetin showed that the length of potato micro-plants of different genotypes is not the same. The longest micro-plants were formed by the ‘VB Meda’ variety (9.03 ± 0.18 cm) and the smallest by ‘VB Venta’ (5.20 ± 0.15 cm) explants. Increases in the length and weight of micro-plants were observed using kinetin at a concentration of 0.8 ml l^− 1.

Genotype

IAR

Kinetin

Micro-plants

Potato

Potatoes can easily adapt to various growing conditions and are distinguished by high yields and nutritional value; therefore, they are very widespread and are considered the most important nanograined agricultural plant in the world. Potatoes are also one of the main raw materials for starch extraction, and the starch content of potatoes is usually between 9% and 25% (Acharjee et al. 2018). Because potato tubers cannot be stored for a long time, the use of potatoes in the form of starch and modified products has become an excellent alternative (Fernie and Willmitzer 2001; Sonnewald and Sonnewald 2014). According to the Food and Agriculture Organization of the United Nations (FAO), potatoes rank fourth after corn, rice and wheat in terms of the volume of world production in tones (FAO 2011). Due to the nutritional value of potatoes since ancient times, they are called the second bread. Due to their excellent taste and nutritional and culinary qualities, potatoes have become a product of almost daily use. Potatoes also have healing properties (Leonel et al. 2017).

The meristematic method of potato seeding is currently the most promising and fastest for seed potato reproduction. Meristem culture may have been the first biotechnological method used to remove viruses from systematically infected potato clones. Over the years, this technique has been successfully combined with the method of microproduction to bring out potato seeds without diseases. The rapid reproduction of these disease-free clones using microproduction in combination with conventional propagation methods has now become an integral part of seed production in many countries (Barrell et al. 2013). The technique of plant tissue culture has become an alternative in recent years and has become popular as an alternative method of vegetative propagation of plants (Hajare et al. 2021). As a modern technology, plant tissue culture has great potential to meet the ever-growing demand of the world. Healthy, thermotherapeutically cleaned of viruses, without bacterial and fungal beginnings, potatoes are grown from the meristematic cells of the seedling growth cone. In this way, not only is good quality achieved, but conditions are created for the significantly faster reproduction of potatoes of new varieties (Roosher 1988; Hajare et al. 2021).

Microdescence is a method of vegetative propagation in culture in vitro designed to allow plants to multiply quickly and in large quantities. When propagated in this way, a large number of plants in a small space are regenerated from a small explant. In this undergraduate work, microvegetative reproduction was used. This method is aimed at cloning plants while maintaining their genetic and physiological characteristics. The optimal composition of the medium and the growing conditions for each species may be fundamentally different, so they are determined experimentally.

Phytohormones are needed to control in vitro plant cells, tissues, organs or entire plant crops according to the desired results: maintain vitality, grow, initiate organogenesis (the formation of organs, for example, the formation of a stem with leaves or roots), and so on. Without phytohormones, plant growth cannot be controlled (Ohnuma et al. 2020; Hajare et al. 2021).

The purpose of the work. To study the effect of growth regulators on micro-plants of potatoes (Solanum tuberosum L.) in vitro.

The experiment was prepared and carried out at the Biotechnology Laboratory of the Lithuanian Center for Agrarian and Forest Sciences and at the Institute of Agriculture of Vokė branch (Vilnius) in 2021.

Microgrowths of edible potatoes (Solanum tuberosum L.) of different varieties (Table 1) of different ripening times: very early 'VB Venta', early 'Goda', medium 'VB Meda' and very late 'VB Aista’, as well as growth regulators IAR (indolyl-3 acetic acid) and kinetin (6-furfurylaminopurin).

Murashige & Skoog (MS) medium (Murashige and Skoog 1962; Saker et al. 2012) is one of the most widely used nourishing media for tissue cultures. An important feature of this medium is the high concentration of nitrates, potassium and ammonium ions. The nourishing medium is sterile in an autoclave at 120°C at 0.75–1 atm. for pressure, 30 min with a pH = 5.7–5.8. Microgrowths were cut and planted in a nutrient medium with a certain concentration of phytohormones (5 ml). The planted explants were kept in vitro in a tumor, where the photoperiod is automatically regulated; during the day for 16 hours, the seedlings grow in the light (12 thousand km). Lx), 8 a.m. in the dark. The air temperature during the day is up to 24°C; at night, it is approximately 21°C, and the humidity is 86–90%. Length (cm) and mass (g) of micro-plants measured during the tests

2.1 The influence of IAR on microgrowths − 2-factor (AxB) test: A – explantants of potato varieties ('VB Venta', 'Goda', 'VB Meda' and 'VB Aista'); B – 3-indolylacetic acid (IAR) concentrations of 0.0; 1.0; 1.25; 1.5; 1.75- and 2.0-ml l^− 1 (solutions prepared by dilution method). Potato explants were planted in media with different concentrations of gold on January 5–7 of this year. Biometric indicators were measured 4 weeks later (Fig. 5), when microgrowths formed new 4–7 leaflets. For one option, 50 pieces were planted. explants, during the entire test – 1200 pcs.
2.2 Effect of kinetins on microgrowths − 2-factor (AxB) test: A – explantants of potato varieties ('VB Venta', 'Goda', 'VB Meda' and 'VB Aista'); B = kinetin concentrations – 0.0; 0.4, 0.5, 0.6; 0.7 and 0.8 ml l^− 1 (solutions prepared by the dilution method). Potato explants were planted in media of different kinetin concentrations on February 9–10 this year. Biometric indicators were measured 4 weeks later, when microgrowths formed new 4–7 leaflets. For one option, 50 pieces were planted. explants, during the entire test – 1200 pcs.

Statistics of Fig. 1–4 show means and their standard deviations (х ± SD). The reliability of differences between samplings was assessed by dispersion analysis with further evaluation of the least significant difference (LSD) using the Statistica 5 software package.

The effect of all phytohormones on plants is quite polyvalent. All of them affect cell growth and division, adaptation and aging processes, substance circulation, respiration, nucleic acid and protein synthesis, etc. However, each group has its own specific effect (Ohnuma et al. 2020). Potatoes, like other plants, have a whole known group of phytohormones. For example, the different hormones contained in potatoes (IAA, CK, OR, JA) stimulate some stages of tuberization, and GA inhibits them (Kolachevskaya et al. 2018). Auxins play a very important role at almost all stages of plant development, which require changes in the meristem. Studies using auxins in vitro highlight their importance for tubers (Roumeliotis et al. 2012).

High concentrations of auxins can and without cytokinins cause mitosis in somatic plant cells. In general, it is necessary to determine the adequate concentrations of auxins and cytokinins for plant regeneration.

3.1 Influence of auxin (IAR) on the formation of micro-plants - The study used 4 potato genotypes, as the in vitro productivity of explants depends on the amount of IAR in the culture medium. Tested nourishing medium with IAR concentrations of 1.0, 1.25, and 1.5 and IAR concentrations of 1.75 and 2.0 ml l^− 1.

The results of the studies showed that the highest microgrowths were the VB Meda varieties (10. 03 ± 0.96 cm), the lowest – 'VB Venta' (6. 2 ± 0.75 cm) (Fig. 1, a). Most of the masses of micro-plants were formed by the varieties of 'VB Meda' (0. 218 ± 0.09 g) explants (Fig. 1, b).

Summing up the results of the length and mass of micro-plants, it can be said that for the explants of the VB Meda variety, the effect of IAR was the greatest. The explants of the 'VB Aista', 'Goda' and 'VB Venta' varieties also developed well.

When using a golden concentration of 1.75 ml l^− 1, a sharp increase in the length of the micro-plants LSD_01(AxB) = 1.430 (Fig. 2, a) and the mass LSD_01(AxB) = 0.047 (Fig. 2, b) is observed.

Summarizing the results of the length and mass of micro-plants, it can be said that 1.0; 1.25; 1.5; The concentrations of the L^− 1 IAR compound of 1.75 and 2.0 ml had a positive effect on most tests. The scientific literature states that for stem morphogenesis, the optimal concentration of auxins is 1.0 ml l^− 1 (Ohnuma et al., 2020). Our study showed that the optimal concentration for microgrowths is 1.75 ml l^− 1. Auxin (IAR) stimulated cell swelling and growth, in part, and cell division.

3.2 The influence of kinetin on the formation of micro-plants - In this study, 4 potato genotypes were used, as the in vitro productivity of explants depends on the amount of kinetins in the culture medium. Tested nourishing medium with 0.4, 0.5, and 0.6 at concentrations of 0.7 and 0.8 ml of l^− 1 kinetin.

The results of the test showed that the length of micro-plants in different potato genotypes was different (Fig. 3, a). The longest microgrowths were shaped by 'VB Meda' (9. 03 ± 0.18 cm) and the smallest ones are exhibitors of the 'VB Venta' variety (5.2 ± 0.15 cm).

Summarizing the results of the length and mass of micro-plants (Fig. 3, b), it can be said that the kinetin effect was greatest on the explants of the ‘VB Meda’ variety. The explants of the 'Goda', 'VB Aista' and 'VB Venta' varieties also developed well.

When using a concentration of kinetin 0.8 ml l^− 1, an increase in the length of the microplants LSD_01(AxB) = 1.286 (Fig. 4, a) and the mass LSD_01(AxB) = 0.021 (Fig. 4, b) were observed. Summarizing the results of the length and mass of micro-plants, it can be said that the effect of 0.4, 0.5, 0.6, 0.7- and 0.8-ml l^− 1 of the kinetin compound concentrations of 0.4, 0.5, 0.6, 0.7- and 0.8-ml l^− 1 on most tests was positive.

It is believed that the formation and development of leaves in vitro had a significant positive influence on the IAR and kinetin. Perhaps the amount of endogenous phytohormones was determined by the genotype of the plant and thus influenced the dependence of organogenesis on the content of exogenous hormones. In the scientific literature, it is indicated that most often the effect of one phytohormone on the metabolism of another is not direct but is a consequence of the functional effect on the activity of tissues.

The author declare that he have no conflicts of interest.

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Table 1 Pedigree information of Lithuanian potato cultivars (Vilnius, 2016-2021)

Characters	Potato varieties
Characters	‘VB Venta’	‘Goda’	‘VB Meda’	‘VB Aista’
Parentage	‘Priekuliu visagrie’ × ‘Pirmūnės’	‘Ausonia’ x ‘Franzi’	‘Matilda’ × No.3093	No.263 x No.476-9
Registration EU	2009	2004	2019	2006
Maturity	Very early	Early	Medium	Very late
Tuber: color of skin	Yellow	Yellow	Yellow	Yellow
Tuber: color of flesh	Light yellow	Light yellow	Yellow	White
Cooking type	A	BC	BC	BC
Taste points	8.0 ± 0.09^*	8.6 ± 0.08	8.2 ± 0.07	7.2 ± 0.05
Yield, t ha^-1	29.9 ± 0.88	37.2 ± 1.21	35.8 ± 1.07	30.4 ± 0.84
Starch, %	15.2 ± 0.65	17.5 ± 0.61	17.4 ± 0.68	20.6 ± 0.97
Dry matter, %	20.5 ± 0.74	21.2 ± 0.49	21.5 ± 0.62	24.9 ± 0.84

Key: cooking type: A - firm, B - fairly firm and C – floury; * - ± SE (standard eror)

The effect of growth regulators on the formation of Solanum tuberosum L. micro-plants in vitro

Status:

Version 1

Abstract

Figures

1 Introduction

2 Materials and methods

3 Results and discussion

Declarations

References

Table 1

Status:

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