Achievements in Genetic Engineering of Amaranthus L. Representatives

Despite the fact that in the modern world more than a thousand edible plants are used for food, only 3 staple cereal crops are grown worldwide: wheat, rice, and maize. Growing a limited number of crops often causes many problems: ranging from the loss of biodiversity, due to the constant cultivation of the same monocultures in the same areas, to the deterioration of soil quality. A way out of this situation is the selection of new untraditional and neglected plants that could grow in a wide range of temperatures, produce high yields and at the same time have a balanced amino acid composition. Pseudocereals of the genus Amaranthus L. meet these criteria. Amaranth grain and plant raw materials are used in many industries: food, medicine, cosmetics. Modern technologies do not stand still. Along with traditional methods of plant breeding, the rapid pace of development involves genetic engineering of plants, which allows the process of creating improved plants to be speeded up several times. The purpose of this study is to analyze and systematize the achievements in the field of regeneration and genetic transformation of representatives of the Amaranthus genus. The results can be used for a practical application: the genetic transformation of species of the genus Amaranthus and other close genera of plants. ARTICLE HISTORY Received: Apr. 12, 2021 Revised: June, 07 2021 Accepted: June 10, 2021


INTRODUCTION
Amaranth is a high-yielding plant. From 1 plant it is possible to obtain more than 5,000 seeds. Moreover, amaranth has a uniquely balanced amino acid composition that ensures easy digestion. Amaranth is a rich source of protein and essential amino acids, deficits of which cannot be compensated by traditional agricultural crops.
Due to the fact that amaranths are indifferent to the type of soil and are drought-resistant, they are grown as a grain crop in countries with a temperate climate (Western Europe), as well as in hot-climate countries, where many traditional crops grow poorly: Mexico, the USA, African countries, India.
Given that amaranth is one of the main food crops in India and Africa, has a unique rich amino acid composition with a high nutritional value, and can serve as a source of biologically active substances for further use in medicine, amaranth plants have undergone improvements for many decades using hybridization, selection and mutagenesis methods.
In recent years, the chemical composition of plants and some agronomic properties have begun to improve using biotechnological methods, namely genetic engineering. Genetic engineering methods make it possible to improve not only the useful properties of a plant, but also to provide additional useful characteristics during plant transformation.
Since it is known that the percentage of Agrobacterium -mediated transformation of plants is often low, usually even before this transformation possible ways of obtaining a large number of transformed plants from a single parent plant are consequently worked out. One of the optimal methods of rapidly increasing the number of plants is considered to be direct regeneration of plants in vitro conditions. Therefore, we first consider the main achievements related to obtaining regenerants of amaranths in vitro.

ACHIEVEMENTS IN REGENERATION OF AMARANTHUS L. SPECIES
To date, there have been many studies on the regeneration and callus formation of amaranth. Basically, the researchers who obtained calluses, had as primary objective their use as a source of secondary metabolites and other valuable substances. In this connection, the largest number of studies devoted to amaranths have had a biochemical orientation.
Amin and colleagues verified the possibility of obtaining the Amaranthus gangeticus L. callus. The leaves, stems and roots were used as initial explants. The scientists observed the formation of calluses in 99.7% ± 0.2% of explants which were derived from stem calluses on MS medium supplemented with 2.0 mg/l NAA(α-Naphthalene acetic acid) + 1.0 mg/l BA (6benzylaminopurine) (Amin et al., 2015).
The callus formation was observed in 100% of explants, with the exception of two lines of A. caudatus L. and three lines of A. cruentus L. and A. hypochondriacus L.. Different concentrations of NAA + BA did not induce callus formation on the A. caudatus explants line AMES5461, while 5.4 μM NAA + 13.3 μM BA caused callus formation only in 43% of PI490458 A. caudatus L. explants. A. cruentus L. lines formed calluses in percentage ratios of less than 100%: AMES2247, 71% on MS medium, with addition of 5.4 μM NAA + 4.4 μM BA; PI511731, 60% on MS medium with addition of 2,4-D + KIN and 67% on MS medium with addition of 5.4 μM NAA + 13.3 μM BA; PI477913 -75% on 2,4-D + KIN and 79% on MS medium with addition of 5.4 μM NAA + 4.4 μM BA.
Plant regenerants were obtained for A. hybridus L. (line 1047) and for A. hypochondriacus L. (line 674). The rate of regeneration was low -8.5% (A. hybridus L.) and 14.3% (A. hypochondriacus L.). Regenerants were also obtained for A. hybridus L., A. hypochondriacus L., A. cruentus L. on MS medium with addition of 2.7 μM NAA + 2.5 μM 2iP (N 6 -(2-isopentenyl)adenine), 2.7 μM NAA + 2.3 μM KIN. The regenerants of A. cruentus L. line 434 and 1034 were obtained on MS medium with addition of 2.7 μM NAA + 4.4 μM BA. The general conclusion of the authors was as follows: the absolute majority of species and lines of amaranths are able to form calluses on most media tested by the authors (almost 100% of callus formation). There was no clear connection between regeneration of shoots and the use of growth regulators. This is due to the strong influence of the genotype of plants on organogenesis. Amaranths have high levels of cytokinins (auxins), which inhibit regeneration processes. The authors believe that the best stimulator of amaranth regeneration was BA.
Mousumi Biswas and colleagues conducted experiments aimed at obtaining calluses for further isolation of betacianins from them (Biswas et al., 2013). The biggest volumes of callus synthesizing betacianins were obtained from explants of stem origin on MS medium supplemented by NAA (0.25 mg/l) + BA (2 mg/l). In addition, researchers found red-purple amaranthine pigment in the callus lines, 2 new yellow pigments and 18 other biologically active phenylpropanoids. A new betaxanthin has been identified and a methyl derivative of arginine betaxanthin was also identified. Pigments were purified by size exclusion chromatography (Biswas et al., 2013).
Flores and colleagues studied the formation of callus and regeneration for the A. hypochondriacus L., A. cruentus L. and A. tricholor L. species. They observed a rapid growth of calluses and abnormal roots on A. hypochondriacus L. and A. cruentus L. leaf disks on MS medium in the presence of 0.1-1.0 mg/l of 2.4-D. At higher levels (1.0-10.0 mg/l) of 2,4-D, embryo-like structures formed from the surfaces and veins of the leaf discs. Shoots were formed from hypocotyl derivative callus on the medium B5 + 0.1 mg/l NAA and 0.1-1.0 mg/l ZEA (zeatin). Lower ratios of ZEA/NAA stimulated the formation of roots from hypocotyl segments (Flores et al., 1982).
Gajdošová, with a team of researchers, selected the ideal conditions for the regeneration and cultivation of Amaranthus cruentus L. 'Ficha' and Amaranthus hybridus (Gajdosova et al., 2007;Gajdosova et al., 2013) 'K-433'. As explants, they used epicotyls with the first pair of leaves, hypocotyls, roots and segments of the leaves of 10-day seedlings. For both species studied, the most effective media for direct regeneration from epicotyls were MS30, supplemented with 5 mg/l BA + 0.01 mg/l NAA, MS30 supplemented with 1 mg/l TDZ (thidiazuron), MS30 supplemented with 3mg/l TDZ + 0.01 mg/l NAA. The most effective medium for induction of callus was MS30 with 6 mg/l NAA + 0.1 mg/l BA (for Amaranthus cruentus L. 'Ficha') and MS30 + 2 mg/l 2.4 D + 0.5 mg/l BAP (for Amaranthus hybridus L. "K-433"). The authors made the following conclusions: in order to obtain regenerants, it is necessary to use mediums with a high cytokinin content: auxins; amaranths are characterized by a high callus forming ability, almost 100% on all tested mediums; regenerants were obtained only from epicotyl segments; the ability to regenerate strongly depends on the genotype, age of plants and used types of explants; the overall regeneration frequency was low (Gajdosova et al., 2007;Gajdosova et al., 2013).
Flores and colleagues investigated the regeneration ability and the callus formation of the following species: A. hypohondriacus L., A. cruentus L., A. tricolor L.. Parts of the hypocotyls were used as explants. The regeneration was indirect (first, callus tissue was obtained). The scientists concluded that the optimal medium for regeneration is B5 supplemented with 0.1mg/l NAA + 0.1-1.0 mg/l ZEA. The callus tissue was obtained from leaf discs of A. hypohondriacus L. and A. cruentus L. Intensive growth of the callus was observed on MS30 medium with 0.1-1 mg/l 2,4-D. However, after addition to the MS30 medium of 0.2 mg/l BA + 2 mg/l NAA and 10% coconut water, they observed shoot induction from callus tissue (Flores & Teutonico, 1986).
The team of researchers headed by Bennici intended to obtain regenerants for the following species: A. hypohondriacus L., A. cruentus L., A. hybridus L., A. caudatus L. As explants, hypocotyls were used. Regeneration was obtained for 2 species as a result: A. hypochondriacus L. (MS30 + 3 mg/l BA + 1 mg/l NAA), A. caudatus L. (MS30 + 3 mg/l KIN + 0.3 mg/l IAA (indole-3-acetic acid). The percentage of regeneration was low (26%). At the same time as the main objective of obtaining regenerants, researchers obtained a callus tisssue. Rapid and intensive callus formation from hypocotyl explants was observed for A. cruentus L. (6 mg/l NAA + 0.1 mg/l BA) and A. hybridus L. (6 mg/l 2.4-D + 0.1 mg/l KIN (Bennici et al., 1992). Arya and colleagues chose A. paniculatus L. as an object of research. Parts of the inflorescence were used as explants. When transferring the explants on the MS30 medium with 8-15 mg/l KIN or MS30 + 5-10 mg/l BA, secondary inflorescences were formed from stems and leaves of the primary inflorescence buds (Arya et al., 1993). Bui van Le and colleagues received regenerants of A. edulis L. from thin cell layers. For experiments, they used thin slices (0.2-0.4 mm) of cotyledons, hypocotyls, roots, tissues from the apical and sub-apical areas. Explants were obtained from 7-day seedlings (Bui van Le et al., 1998). Regenerants were obtained solely from tissues taken from the apical and sub-apical zone. Only callus tissue was obtained from all other types of explants.
A team of researchers headed by Bagga, studied the regeneration ability and callus formation of A. paniculatus L. The hypocotyls were used as the explants. Regeneration of 1-2 shoots from one end of the hypocotyls explants was obtained on medium B5 + 1ppm KIN + 1 ppm NAA; on medium B5 + 0.5 mg/l KIN + 0.1 mg/l NAA numerous buds formed (10-14 pieces), from which stems developed later. Intensive callus growth was observed on medium B5 + 1 mg/l GA3 (gibberellic acid) + 1 mg/l KIN + 1mg/l 2,4-D (Bagga et al., 1987).
Jofre-Garfias and co-authors obtained embryos from the cotyledons of A. hypochondriacus L. cv. Azteca on medium MS3 + 10% coconut milk and MS3 + 10μM 2.4-D (Jofre-Garfias et al., 1997). Pal and colleagues obtained A tricolor regenerants from hypocotyls and epicotyls of 7-day seedlings on MS30 + 13.2 μM BA +1.8 μM NAA . In another study, Pal argued that he and his colleagues received regenerants of A. spinosus from the culture of "hairy" roots. Regenerants were obtained on MS30 medium without growth regulators (spontaneous regeneration) and on MS30 medium + 2 mg/l ZEA .
Swain and his colleagues obtained A. tricolor regenerants from the culture of "hairy" roots. Regenerants were obtained (on MS30 medium without growth regulators (spontaneous regeneration) and on MS30 medium + 2 mg/l ZEA (Swain et al., 2009;Swain et al. 2010).

ACHIEVEMENTS IN THE TRANSFORMATION OF AMARANTHUS SPECIES AND FUTURE PROSPECTS The next step after obtaining regenerated plants is genetic transformation. The number of studies devoted to genetic transformation of Amaranthus is rather small.
So far, it is reported that genetically transformed parts or whole plants of amaranth have been obtained by two different methods: Agrobacterium-mediated transformation and transformation using the "floral-dip" method.
The Agrobacterium -mediated transformation method was developed on the basis of a natural process. Wild soil bacterium Agrobacterium rhizogenes or tumefaciens is able to infect plants, causing the appearance of "hairy" roots (A. rhizogenes) or tumors -crown galls (A. tumefaciens). At the same time as the infection process, the transfer and integration of two groups of genes into the plant genome occurs. Genetically modified Agrobacterium transfers the genes of interest or selective genes needed by humans into the plant's genome.
The first experiments on the transformation of amaranths were unsuccessful (De Cleene & De Ley, 1976). At present, it has been proved that transgenic amaranth plants can be obtained through Agrobacterium-mediated transformation. But still there are very few studies devoted to amaranth transformation.
There is no information on the transformation of A. caudatus, varieties of which are also used in agriculture.
Transgenic roots were obtained for A. tricolor L. plants by Swain and colleagues (Swain et al., 2010) and for A. spinosus L. by Pal and colleagues . The transformation of amaranths was carried out using a wild strain of Agrobacterium rhizogenesis A4. Research group Taipova, Kulaev and others obtained transgenic roots for A. cruenthus L. from epicotil segments .
Positive results were also obtained in the transformation of amaranth species using strains of Agrobacterium tumefaciens. Jofre-Garfias and co-authors transformed the Azteca variety of A. hypochondriacus L. They used the vector from Agrobacterium tumefaciens with marker genes (Jofre -Garfias et al., 1997). Transgenic A. tricholor L. was obtained by two different groups of scientists -Swain and colleagues and Pal with co-authors (Swain et al., 2009;. A team of researchers headed by Pal used a vector with marker genes. Taipova and Kulaev obtained regenerated transformed plants from epicotil explants after Agrobacterium-mediated transformation Taipova & Kuluev, 2018).
There are also 3 studies devoted to amaranth transformation through inflorescences by the "floral-dip" method -Umaiyal Munusamy and co-authors. They used a vector with selective genes (Munusamy et al., 2013).

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Thus, at the moment, transgenic amaranth plants have been already obtained with selective genes, marker genes and genes of interest. Research into the transformation of amaranth continues. In the near future, transgenic amaranths may appear that have an improved biochemical composition and new useful properties.

CONCLUSION
Amaranth is unique plant. Its nutritional value and optimal amino acid composition have already been evaluated in many countries around the world. In Western Europe, the plant has already gained popularity and it is possible find products with amaranth on the shelves of supermarkets. In Ukraine, we also have a small range of products that include amaranth.
As can be understood from our previous experimental work and the work of other authors, there are difficulties in achieving regeneration for many species of amaranths. If regenerants are obtained, the percentage of regeneration does not exceed 30 percent, which is clearly not enough for further obtaining transformed plants after agrobacterial transformation.
Therefore, other transformation techniques are being developed, for which it is not necessary to obtain regenerated plants. The alternative transformation method is called "floral-dip'. According to published studies, transformed plants have been obtained using this method.
At present, mainly transgenic amaranth plants have been obtained, which were transformed by agrobacteria that carried vectors containing selective genes. Only one group of authors obtained transgenic plants with not only selective genes, but also genes of interest.
In the near future, a greater number of amaranth species will be obtained, which will present additional useful features, such as, for example, protein synthesis, which can be used in medicine. The authors hope, that in the near future, amaranth will achieve the position of a recognized niche of the food and medicine industries.

Declaration of Conflicting Interests and Ethics
The author declares no conflict of interest. This research study complies with research and publishing ethics. The scientific and legal responsibility for manuscripts published in IJSM belongs to the author.