Waldemar Kiszczak

Red raspberry ( Rubus idaeus L.) is a small-fruit crop of key economic importance, widely cultivated for fresh consumption and processing, and valued in traditional medicine. Vegetatively propagated material is subject to increased pathogen pressure, especially viruses, viroids, and phytoplasmas, leading to growth suppression, yield losses of up to 50%, and heightened susceptibility to secondary infections. Ensuring healthy, pathogen-free planting stock is therefore a priority in production and breeding. This review synthesizes current knowledge on cryopreservation methods applicable to raspberry, with particular emphasis on their suitability for virus eradication (shoot-tip cryotherapy) and identifies the technical parameters that determine procedural success. Cryopreservation and cryotherapy protocols based on vitrification (including variants based on plant solution vitrification and droplet vitrification) and encapsulation-dehydration were analyzed. The focus was on shoot apical meristems as the starting material and on combining cryotherapy with other sanitation procedures, for example thermotherapy. Among the available approaches, the highest post-thaw regeneration rates were obtained with vitrification protocols and with encapsulation-dehydration. Efficiency increases when cryotherapy is combined with thermotherapy. Success is strongly influenced by explant size and physiological state, preculture conditions (for example sucrose concentration), cryoprotectant composition and exposure time, and cooling and warming profiles. Beyond its role in the long-term conservation of genetic resources, cryopreservation is an effective tool for sanitizing raspberry material. Shoot-tip cryotherapy enables the production of virus-free plants, which is crucial for establishing healthy plantations and breeding programs. Incorporating standardized protocols into nursery certification systems may accelerate the dissemination of clean stock and, in the long term, reduce production losses. The evidence reviewed confirms that optimized cryopreservation protocols provide a reliable framework for both long-term conservation of germplasm and the regeneration of healthy raspberry planting material.

The process was examined or the effect of culture conditions on in vitro gynogenesis in red beet was analyzed, conditions were modified or optimized. A significant influence of the genotype on the gynogenesis process was demonstrated. Of the eight genotypes, 58.3% planted ovules regenerated embryo-like structures in breeding line 411, 2.1% in RA-10, RA-11, RA-12 breeding lines and 0.9% embryo-like structures in Opolski. For the gynogenesis induction, B5 medium containing 0.1 mg L–1 2,4-dichlorophenoxyacetic acid was the most effective from all tested media. On this medium, the highest number of gynogenetic embryo-like structures was obtained. Most of the plants were regenerated on MS medium supplemented with 30 g L–1 sucrose, 0.2 mg L–1 6-benzylaminopurine and 1 mg L–1 indole-3-acetic acid. Thirty nine percent of regenerated plants acclimatized. Cytometric evaluation of gynogenetic plants of four tested genotypes revealed that in three genotypes, 100% of tested plants were haploid. Plants showed diploid ploidy level in one genotype. Isoenzymatic analysis of gynogenetic plants demonstrated that 95% and 70% of examined populations were homozygotic for the phosphohexose isomerase isoenzyme and the aspartato aminotransferase isoenzyme, respectively. During the next generation sequencing, 93% of reads were successfully mapped, from which 83% to 85% were mapped in pairs. For 15% of pairs it was clear that obtained sequence was fully homozygous, the rest of the readings were not unambiguous, but similar to the sequence of a homozygous base pair system.

Conditions of in vitro gynogenesis process in red beet was examined. A significant influence of the genotype on the gynogenesis process was demonstrated. Of the eight genotypes, 58.3% planted ovules regenerated embryo-like structures in breeding line 411, 2.1% in RA-10, RA-11, RA-12 breeding lines and 0.9% embryo-like structures in ‘Opolski’. For the gynogenesis induction, B5 medium containing 0.1 mgl^-1 2,4-dichlorophenoxyacetic acid was the most effective from all tested media. On this medium, the highest number of gynogenetic embryo-like structures was obtained. Most of the plants were regenerated on MS medium supplemented with 30 g l^-1 sucrose, 0.2 mg l^-1 6-benzylaminopurine and 1 mg l^-1 indole-3-acetic acid. Thirty nine percent of the regenerated plants acclimatized. Cytometric evaluation of the gynogenetic plants of four tested genotypes revealed that in three genotypes, 100% of tested plants were haploid. Plants showed diploid ploidy level in one genotype. Isoenzymatic analysis of gynogenetic plants demonstrated that 95% and 70% of examined populations were homozygotic for the phosphohexose isomerase isoenzyme and the aspartato aminotransferase isoenzyme, respectively. During the next generation sequencing, 93% of reads were successfully mapped, from which 83–85% were mapped in pairs. For 15% of pairs it was clear that the obtained sequence was fully homozygous, the rest of the readings were not unambiguous, but similar to the sequence of a homozygous base pair system.

The induction of haploid cell development into normal plants enables the production of doubled haploid lines, which are homozygous and can be used in breeding programs as an alternative to conventionally derived inbred lines. In this paper, we present the historical background and current status of the attempts of haploid induction in carrot (Daucus carota L.). Economically, carrot is one of the most important vegetables. It is an outcrossing diploid (2n = 2x = 18) species. Nowadays, the seeds of hybrid cultivars constitute the majority of the carrot seeds sold in the world. Hybrid cultivars of carrot are produced using inbred populations. Inbreeding in this species is difficult due to an inbreeding depression and is also time-consuming, as it is a biennial crop. Therefore, the implementation of the haploidization technology into the breeding programs of carrot is of high interest. Androgenesis, gynogenesis and induced parthenogenesis are the methods that have been used for haploid induction, and their potential in haploidization of carrot is discussed. The centromere-specific histone 3 variant (CENH3) and its manipulation in carrot is also acknowledged.

The influence of polyamines (PAs), putrescine (Put) and spermidine (Spd) on the efficiency of gynogenesis in ovule cultures of red beet (syn. beetroot) (Beta vulgaris L. vulgaris) cultivar “Czerwona Kula” and breeding accessions no. 3/2010 and no. 7/2008 was investigated. The effect of Put on the process of plant regeneration from gynogenetic embryos was studied. The response to the applied PAs was strongly dependent on the genotype. In “Czerwona Kula”, an increase in the number of obtained embryos was achieved by using each of the two PAs in the B5 medium. The effect of Spd was stronger. Put added to the regeneration medium at the concentration of 0.5 mg L⁻¹ increased the number of obtained plants. All shoots placed on the rooting medium supplemented with 160 mg L⁻¹ Put formed roots. The distribution of ploidy and homozygosity of gynogenetic plants depended on the genotype. Of the tested genotypes, the highest number of haploid plants, 68%, was obtained in red beet “Czerwona Kula”. The highest percentage of homozygotes, 69% for the glucose phosphate isomerase (GPI, E.C.5.3.1.9) isoenzyme and 100% for the aspartate aminotransferase (AAT, E.C.2.6.1.1) isoenzyme, was obtained in the population of gynogenetic plants of cultivar “Czerwona Kula”.

Yacon becomes an important plant with strong medical and dietary potential. Therefore, studies on the optimization of yacon micropropagation were undertaken. Reproduction of yacon is conducted by traditional methods with green seedlings or long offsets taken from the underground and aboveground rootstock. The influence of different disinfection methods, various initial explants and growth regulators, their concentrations, and combinations on the efficiency of micropropagation have been analyzed. The most effective disinfection was the use of 70% ethanol and 0.5% Tween 20. The optimal initial explants were apical and lateral buds cultured on Murashige and Skoog (MS) medium with 0.2 mg L–1 kinetin and 1 mg L–1 indole-3-acetic acid. The most intensive shoot regeneration occurred on MS medium with 1 mg L–1 thidiazuron and 1-naphthylacetic acid. Histological observations demonstrated the strong effect of growth substances on shoot organogenesis from callus tissue. Shoots obtained from in vitro cultures rooted easily on modified MS media with ½ or ¼ concentration of macroelements, 20 g L–1 sucrose and 3 mg L–1 1-naphthylacetic acid. All plants adapted to ex vitro conditions.

Hybrid varieties dominate the red beet market. The breeding process necessary to produce these cultivars is very difficult and time consuming. The application of in vitro gynogenesis can reduce the time needed to produce the corresponding homozygous pure lines to a few months. Our research team has developed a method to obtain red beet doubled haploid plants by gynogenesis. The best medium for gynogenesis induction is the B5 medium with the addition of 0.5 mg/L IAA, 0.2 mg/L BA, and 322 mg/L putrescine, whereas the best medium for shoot induction from embryos proved to be the MS medium supplemented with 0.1 mg/L NAA, 0.1 mg/L BA, and 0.5 mg/L putrescine. The shoots obtained were rooted on MS medium containing half the concentration of microelements and 3 mg/L NAA, 160 mg/L putrescine, and 20 g/L sucrose. Ploidy evaluation of gynogenetic plants was performed by flow cytometry and homozygosity or heterozygosity was determined by two isoenzymatic systems: PGI and AAT.

Carrot is a vegetable of increasing economic importance. New hybrid cultivars are constantly required to meet the changing market needs. The application of anther culture significantly shortens the difficult and long-lasting breeding of carrot. We examined all the stages of the process of generating androgenic plants: induction of embryos in anther cultures, regeneration and acclimatization of produced plants, their evaluation, ploidy and homozygosity, and many other factors affecting their effectiveness. Every factor has been optimized by experimentally selecting the optimal level. As a result, a full protocol of producing homozygous plants using anther cultures was developed, which is presented in this chapter.

Experiments were made in order to examin the influence of various factors on the induction of androgenesis in heterozygous breeding material of tomato. The factors like: length of buds the manner of sterilization, type and the composition of induction media, genotype and thermal shock were included in conducted experiments. Most of all sterile cultures with the highest number of anther-derived callus were obtain by applying 2,5% calcium hypochlorite for 5 minutes. Anther-derived calli was obtained in 9 breeding lines from 18 used genotypes. The induction medium with the composition based on B5 medium with the addition of 750 mg L-1 calcium chloride and 100 g L-1 sucrose proved to be the best for inducing androgenesis. The addition of thidiazuron and NAA to this medium in the following season and silver nitrate in the other experiminet improved the efficiency of this process, which was depended on the genotype. Despite the lack of statistically significant differences, the highest number of anther-derived calli was obtained when anthers were cooled for 2 days in +4oC in the refrigerating chamber.

Background and Objective: Currently, an interest in the cultivation of yacon is growing, which is a plant with strong medical potential. Therefore, studies on the optimization of the yacon micropropagation method in tissue cultures were undertaken. Materials and Methods: Reproduction of this plant is conducted with the use of traditional methods by green seedlings or 8-12 cm long offsets (‘seeds’) taken from the underground and aboveground rootstock (‘crown’). The influence of different disinfection methods, various initial explants and growth regulators, also their concentrations on the efficiency of micropropagation protocol in conducted research was analyzed. Results: The most effective disinfection was the application of 70% ethanol and 0.5% Tween 20. The optimal initial explants in the current research were apical and lateral buds cultured on MS medium supplemented with 0.2 mg L^-1 kinetin and 1 mg L^-1 indole-3-acetic acid. The most intensive shoot regeneration occurred on MS media with the addition of 1 mg L^-1thidiazuron and 1-naphthylacetic acid. Histological observations demonstrated the strong effect of these growth substances on shoots organogenesis from callus tissue. Conclusion: Shoots obtained from in vitro cultures rooted easily on modified MS media with ½ or ¼ concentration of macroelements, 20 g L^-1 sucrose and 3 mg L^-1 1-naphthylacetic acid and then adapted to ex vitro conditions in 100%. APA-7 Style Kiszczak, W., Kowalska, U., Burian, M., Glinska, S., Górecka, K. (2021). Influence of Growth Regulators on Micropropagation System of Medicinal Plant Yacon (Smallanthus sonchifolius (Poepp.) H. Rob.) . Asian Journal of Emerging Research, 3(1), 44-48. https://doi.org/10.3923/ajerpk.2021.44.48 ACS Style Kiszczak, W.; Kowalska, U.; Burian, M.; Glinska, S.; Górecka, K. Influence of Growth Regulators on Micropropagation System of Medicinal Plant Yacon (Smallanthus sonchifolius (Poepp.) H. Rob.) . Asian J. Emerg. Res 2021, 3, 44-48. https://doi.org/10.3923/ajerpk.2021.44.48 AMA Style Kiszczak W, Kowalska U, Burian M, Glinska S, Górecka K. Influence of Growth Regulators on Micropropagation System of Medicinal Plant Yacon (Smallanthus sonchifolius (Poepp.) H. Rob.) . Asian Journal of Emerging Research. 2021; 3(1): 44-48. https://doi.org/10.3923/ajerpk.2021.44.48 Chicago/Turabian Style Kiszczak, Waldemar, Urszula Kowalska, Maria Burian, Sława Glinska, and Krystyna Górecka. 2021. "Influence of Growth Regulators on Micropropagation System of Medicinal Plant Yacon (Smallanthus sonchifolius (Poepp.) H. Rob.) " Asian Journal of Emerging Research 3, no. 1: 44-48. https://doi.org/10.3923/ajerpk.2021.44.48