Morphogenesis of Anthers Isolated From Cabbage (Brassica Oleracea L.) In Vitro

This study aimed to optimize the steps of obtaining regenerated cabbage plants by direct embryogenesis from isolated anthers and ovaries. Stepwise pretreatment of inflorescences was usedfor the studied hybrids and inbred lines. First, the inflorescences were placed in water and kept at a temperature of +4-6∘C for one day without the use of biologically active substances. Then the inflorescences were placed in a solution of the drug Dropp (10 mg/l) and cultivated for two days. After that, the anthers and ovaries were isolated from the flower buds and cultured on the MS culture medium at a temperature of + 32∘C for one day. The cultivation of the isolated explants on a nutrient medium (containing 0.01 mg/lof Dropp, 1.0 mg/lof NAA, 500 mg/lof asparagine, 100 mg/l of tyrosine, and 10 g/l of sucrose)led to an increase in their morphogenetic potential in the culture of anthers and ovaries (by 3.42% and 5.54%, respectively).A cytological method was usedto demonstrate the haploid nature of the regenerating plants. The number of chromosomes in the root meristem andleaves, and the chloroplasts in the closing cells of the stomatawere calculated. Keywords: cabbage, culture in vitro, regenerated plants, anthers, ovaries, reproductive organs

Double Haploid Development and Assessment of Androgenic Competence of Balkan Pepper Core Collection in Bulgaria

This study was designed to assess the androgenic potential of 180 pepper accessions and 11 progenies (four F1 and seven BC) possessing PMMoV resistance in order to complement an ongoing pepper breeding program. The experiment was carried out in 10 replications with 20 anthers for each accession in two different induction mediums from 2017 to 2019. The highest androgenic response was observed in culture medium 17-2 but differences between two mediums were nonsignificant. From a total of 191 genotypes, 102 genotypes expressed a potential for direct embryogenesis. Embryo induction was seen to be genotype-dependent and decreased in the following order: Pumpkin > Conical > Bell or blocky > Round > Elongate as the most responsive genotypes with over 10% reacted anthers being observed in CAPS-23, CAPS-29, CAPS-127, CAPS-157, CAPS-169, F1 and BC 887 derived from CAPS-23. The number of regenerated plants was higher in the conical group and least in the round varietal group. Regenerated plants were examined visually and by flow cytometry for identification of spontaneous doubled haploids (DH) and haploids. Those originating from F1 and BC progenies were additionally evaluated by a CAPS marker targeting L4 allele for resistance against PMMoV. Obtained results revealed two groups consisting of homozygous susceptible and resistant plants. Therefore, use of anther culture in ongoing breeding will greatly facilitate the pepper genetic improvement.

Direct embryogenesis from anther culture of hot chilli Capsicum annuum L.

In Viet Nam, local varieties of chili have a distinctive aroma and pungency. However, the generation of pure lines from pollen culture in local hot chilli has been very limited reported. Therefore, this study was to relate flower bud size with microspore developmental stages and the culture media have concentration changed of growth regulator effects on the in vitro androgenesis. Flower buds were randomly collected and visually divided into three stage based on both petal and sepal size. Then anther was cultured on MS basal medium with different concentration of hormones NAA changed 0.1 - 0.7 mg/L and kinetin changed 1.0 - 3.0 mg/L, BA changed 0.5 - 1.5 mg/L. The results showed that bud flower have anthers are light violet in color, 2.5 mm long, consisted of anthers with 80 % uni-nucleate and 20 % bi-nucleate microspores were selected. In induction culture media, it was observed that MS medium with 2.0 mg/L Kinetin and 0.5 mg/L NAA gave higher embryo frequency. MS medium with 1 mg/L BA is the best medium for embryo germination and inducting shoots. And ½ MS medium for shoot elongation and rooting.

Induction of Embryogenesis in the Culture of Isolated Microspores of Wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L.) haploids and doubled haploids are widely used in breeding, the investigations of a combinative variability and its stabilization in homozygotes. In four domestic varieties of winter wheats (Moskovskaya 56, Moskovskaya 39, Galina, Nemchinovskaya 24) and three domestic varieties of spring wheats (Ester, MIS, Amir). With spring wheat variety Falat as a control, the efficacy of embryogenesis in isolated microspores was tested using standard protocol for induction of direct embryo formation in the isolated microspore culture. In all winter varieties there was shown a low frequency of cytoplasmic strands, which are typical for the embryogenic microspores, whereas in the spring varieties it was high. After 4 days cultivation in the medium used for induction, the microspore viability decreased in winter varieties. and another 10 days later the Viable cells were not observed. The spring varieties developed the multicellular structures, which could produce embryos. The reference variety Falat produced 28 % of proembryoids, able mostly to further embryonic formation. Basing on these results, the protocol for inducing direct embryogenesis in wheat microspores was modified, including maltose concentration in medium, the conditions of spikelet heat treatment, the number of ovaries and time when they were added to the culture, the combination and concentration of hormones in the media for induction and cultivation.

Phloroglucinol Mediated Plant Regeneration of Ornithogalum dubium as the Sole “Hormone-Like Supplement” in Plant Tissue Culture Long-Term Experiments

Tissue culture is an essential requirement in plant science to preserve genetic resources and to expand naturally occurring germplasm. A variety of naturally occurring and synthetic hormones are available to induce the processes of dedifferentiation and redifferentiation. Not all plant material is susceptible to tissue culture, and often complex media and hormone requirements are needed to achieve successful plant propagations. The availability of new hormones or chemicals acting as hormones are critical to the expansion of tissue culture potentials. Phloroglucinol has been shown to have certain hormone-like properties in a variety of studies. Ornithogalum dubium, an important geophyte species, was used to characterise the potential of phloroglucinol as the sole plant-like hormone in a tissue culture experiment. Tissue culture, plant regeneration, total phenolic and genetic variability were established by applying a variety of methods throughout long-term experiments. Phloroglucinol did induce callus formation and plant regeneration when used as the sole supplement in the media at a rate of 37%, thus demonstrating auxin/cytokines-like properties. Callus formation was of 3 types, friable and cellular, hard and compact, and a mixture of the two. The important finding was that direct somatogenesis did occur albeit more frequently on younger tissue, whereby rates of induction were up to 52%. It is concluded that phloroglucinol acts as a “hormone-like” molecule and can trigger direct embryogenesis without callus formation.

Somatic-embryogenesis derived leaf-rust-tolerant clones of arabica coffee to deal with climate change

The high-altitude coffee growing, Arabica, is likely subject to the global warming effect as they are prone to leaf-rust attacks at a higher temperature. It supplied 70% of world coffee production for its popularity concerning its delicacy and aromatic flavor. Utilization of, genetically, superior planting materials, i.e. leaf-rust-tolerant Arabica, has become an essential point, as may provide the potential solution to prevent the lost production due to leaf-rust attack. Andungsari 2K (AS 2K), S795, AS1, and Sigararutang are some of the potential leaf-rust-tolerant Arabica clones in Indonesia. Vegetative propagation by somatic embryogenesis may support the availability of superior plant materials quickly. The major aim of this experiment was to study the effect of different clones on germination step after the preliminary stage of direct-embryogenesis from leaf explants with combinations of medium between auxin (2,4-D) and cytokinin (2-ip). Embryo germination stage where embryoid was transferred to the germination medium consisting of MS medium without hormones. The results revealed that the growth location and texture of callus, as well as growth patterns and colour of embryogenic callus, were significantly influenced through the different combinations of medium and clones. The clone of S795 exhibits the highest embryo germination percentage of up to 100% within 8 weeks experiment period.

Peculiarities of in vitro parthenogenesis of unpollinated maize ovaries

The maize line AT-1 is characterized by a hereditary predisposition to parthenogenesis. The aim of this investigation is to study parthenogenetic embryo development in the culture of unpollinated ovaries in vitro . The unpollinated ovaries were explanted in 1, 3, 5, 7, 10, 15 days after the appearance of stigmas from ears. The nutrient medium included mineral components of MS, vitamins, sucrose (9,0%), 2,4-D (2,0 mg/l), agar-agar. The structure of megagametophytes at the time of inoculation of the ovaries and on the 3rd, 7th, 14th, 21th, 28th day of cultivation was studied. The first divisions of unfertilized egg cells were observed on the 5th-7th day after appearance of stigmas from ears, independently from whether all this time the ovaries were on the mother plant or they were inoculated into the nutrient medium. The formation of the autonomous abnormal endosperm in some cultivated ovaries was detected. The abnormal endosperm disturbed normal development of the proembryo. As a rule, the ovaries with embryo and endosperm degenerated. In the absence of endosperm, the morphogenesis of parthenogenetic proembryos was carried out in one of two directions in vitro : 1) development of plants by direct embryogenesis; 2) regeneration of plants from numerous embryoids, raised on the surface of globular proembryos. The second direction was prevailed. The culture of unpollinated ovaries can be a promising method of mass haploid regenerants not only in maize, but also in other types of agricultural plants.

In vitro Direct Regeneration from Node and Leaf Explants of Phalaenopsis cv. ‘Surabaya’

An efficient and reproducible procedure for the direct regeneration of phalaenopsis cv. ‘Surabaya’ using of nodal explants and leaf segments derived from in vitro flower stalk was conducted. Three experiments were carried out for shoot development and subsequent plant regeneration: Direct shoot regeneration from nodal explants of Phalaenopsis cv. ‘Surabaya’ flower stalks on MS added with different combination of NAA and BAP, direct regeneration of protocormlike bodies (PLBs) from leaf explants in a MS with different concentrations of the TDZ, acclimatization of regenerated plantlets in different mixture of components and nutrients. The results showed that 5 mg/l BAP and 2 mg/l NAA were most effective concentration for shoot regeneration, regenerated shoots were cultured on half strength of MS containing activated charcoal, IAA and NAA at various concentrations, highest number of root (6.7) was obtained in higher concentration of NAA (2 mg/l). TDZ induced a higher frequency of embryogenesis from leaf explants than BAP, the highest number of embryos per explant was 22.45 at 3 mg/l TDZ. Altogether, BAP at higher concentration (10 mg/l) with 1 mg/l NAA had the highest enhancement on the amount of direct embryogenesis. In our investigation 87.20% plantlets via nodal explants survived acclimatization process in medium containing cocopeat and coal (1 : 1). The survival rate of regenerated plantlets via nodal explants (82.07%) was more than of regenerated plantlets via leaf explants (70.47). This protocol provides the basis for further investigation on micropropagation and breeding programs in Phalaenopsis cv. ‘Surabaya’.Plant Tissue Cult. & Biotech. 25(2): 193-205, 2015 (December)