Frequency Plant Regeneration Research Articles

Evaluating the effect of cupric sulphate on morphogenic structure induction and plant regeneration in spring barley anther culture in vitro

Aim. Investigation was aimed to ascertain possibility to increase the efficiency of spring barley haploid production in anther culture in vitro by addition of cupric sulphate to the solution for cut tiller cold pretreatment and by increase of cupric sulphate concentrations in the media for morphogenic structure induction and plant regeneration. Methods. Cut tillers were cold pretreated for 5 days at 4ºC in water and in the solutions contained 5 µM and 10 µM of cupric sulphate. Isolated anthers were cultivated on agar solidified nutrient medium containing N6 macro-and MS micronutrients, physiologically active substances, maltose (9.0 %) and on the media with increased (10 µM) content of cupric sulphate. Results. No effect of cupric sulphate addition on cut tiller viability and morphogenesis in anther culture in vitro was found. The lowest plant regeneration frequency was obtained when cupric sulphate was added both to inductive and regenerative media. Сonclusions. Addition of cupric sulphate to the pretreatment solution as well as increase of this microelement concentration (up to 10 µM) in the nutrient media can’t be considered as the methodological approach suitable to enhance the efficiency of spring barley haploid production in anther culture in vitro.

Development of regenerated plants of interspecific wheat hybrids using immature embryos as explants

Nowadays particular attention in winter wheat breeding is paid to the introduction of biotechnological methods. The culture of immature embryos in vitro is an alternative way to produce hybrids that often do not develop fully in vivo. The purpose of the current study was to estimate the ability of immature embryos of interspecific winter wheat hybrids for callus induction, morphogenesis, and plant regeneration on artificial nutrient media, as well as to select optimal conditions for obtaining full-fledged plants with all the botanical characteristics of the species. The research material included 5 hybrid combinations of interspecific crosses between durum (Triticum durum DESF.) and common (Triticum aestivum L.) winter wheat. The material was collected during one field season (2022). Immature embryos were used as explants. Culture media were according to Murashige-Skoog (MS), and they differed in the content of hormones and organic substances. There has been found that the hybrid combinations were characterized by a variety of morphogenic responses. The manifestation of callusogenesis was not high enough and averaged 22–32 % depending on the nutrient medium. Rhizogenesis averaged from 1–6.7 %, and ended with the formation of roots. The process of formation of embryoid- and hemmorizogenesis was best demonstrated by the hybrid combination No.1, and on two media. All processes of morphogenesis excepting ‘rhizogenesis’ varied greatly among hybrid combinations. The largest number of regenerated plants was obtained from hybrid combination No.1 (61 pcs., 54.5 %). After vernalization and acclimatization, 38 % of the plants survived from the number of planted embryos. The largest number of heads (79 pcs.) was formed according to hybrid combination No.1, on average 1.46 heads per plant. However, not all heads were fertile. The results of the correlation analysis made it possible to identify indicators that closely correlated with the amount of plant regeneration, namely, the high yield of embryoid- and hemmorizogenic structures ensured a high frequency of plant regeneration (r = 0.769).

Optimization of explants, media, plant growth regulators and carbohydrates on callus induction and plant regeneration in Citrus jambhiri Lush.

Callus induction was attempted from the four explants viz. root, cotyledon, epicotyl, and leaf segments excised from in vitro raised seedlings of C. jambhiri. Among various MS media supplementations with growth regulators and carbohydrates, the maximum (95.50%) and the earliest (8.30 days) callogenesis was obtained in epicotyl segments, when cultured on MS medium supplemented with NAA (10.0 mgl-1) + BAP (1.0 mgl-1) + sucrose (8%). The modified MS (macro and micro-nutrients reduced to half) fortified with BAP (5.0 mgl-1) + GA3 (3.0 mgl-1) recorded maximum shoot regeneration (43.10%) from callus, with an average of 5.30 shoots per callus after 35.50 days of culturing. However, prolonged exposure to GA3 resulted in thin elongated shoots and leaves. The age of the callus substantially influenced the plant regeneration frequency. The potency of the callus to regenerate decreased significantly with an increase in the age of the callus. Shoot regeneration was recorded maximum (43.43%) in 60 days old calli, followed by 90 days old (30.48%) calli, whereas it was minimum (10.46%) in 150 days old calli. The maximum (79.50%) shoot proliferation was recorded in MS medium supplemented with BAP (1.0 mgl-1) + Kin (0.5 mgl-1) with an average of 5.06 shoots per culture. The MS medium fortified with NAA (1.0 mgl-1) + IBA (1.0 mgl-1) induced maximum (77.33%) rooting, with an average of 3.19 roots per shoot after 13.4 days of culturing. Rooted plants were hardened and survived the best (83.6%) on the potting mixture consisting of cocopeat + vermiculite + perlite (2:1:1).

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment

AbstractInduced mutations are important for genetic research and breeding. Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations. However, mutations must be fixed prior to phenotyping or field trials, which requires additional rounds of self-pollination. Microspore culture is an effective method to produce double-haploid (DH) plants that are fixed homozygotes. In this study, we conducted ethyl methanesulfonate (EMS)-induced mutagenesis of microspore cultures of barley (Hordeum vulgare) cultivar ‘Hua30’ and landrace ‘HTX’. The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration. The two genotypes showed different regeneration efficiencies. The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations, revealed by whole-genome sequencing, highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants. Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture (somaclonal variation) rather than EMS-induced mutagenesis. In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials, this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations (mainly by fine-tuning the mutagen dosage).

Use of mannitol for cold pretreatment of isolated spikes and as a component of nutrient medium for spring barley anther culture in vitro

Aim. The investigation was aimed to compare the efficiency of mannitol as a component of spike cold pretreatment solution and of medium for spring barley haploid production via anther culture in vitro. Methods. Field-grown plants of two genotypes with a contrast androgenic capacity were used as donors. Cut tillers were pretreated in water at 4⁰C for 5 days, while aseptically isolated spikes were kept in 0.3 M mannitol for 10 days at the same temperature. Isolated anthers were inoculated on inductive media containing N6 macro-, MS micronutrients, organic supplements, maltose (9.0%); the media with and without 0.1M mannitol differed in solidifying agents. Results. In highly-responsive line DH00-126, there was an almost two-fold increase in mmgreen plant regeneration frequency (from 48 to 58 plants per 100 anthers) in all experimental variants in comparison to the control. In recalcitrant variety Mebere, the combination of tiller pretreatment in water with anther cultivation on inductive starch-gelling media supplemented with 0.1 M mannitol resulted in the highest green plant yield. Conclusions. Both methodical approaches were proved to be applicable to increase the efficiency of haploid production in spring barley anther culture in vitro.

High throughput anther culture response in an upland rice cross ‘Khandagiri x Dular’

Aim: The experiment was conducted to maximize anther culture response in an upland indica rice cross amenable for drought tolerance breeding. Methodology: The cold pre-treated anthers of a cross Khandagiri (drought sensitive) x Dular (drought tolerant) were cultured on N6, SK3, MS and CIM media at varying concentration of hormones (2,4-D, Kn and NAA) to assess callusing response. Embryogenic calli were placed on regeneration medium (RM) with varying concentration of Kn, BAP and NAA for plant regeneration. The plantlets were acclimatized in half-strength MS basal liquid medium for one week before transferring to pot mixture (peat moss: perlite 2:1, v/v) for plant establishment in glasshouse under partial shade. Finally, the plantlets were checked for doubled haploid status by cytological study of root tips. Results: Anther culture response was found to be media and genotype-specific. F1-progenies responded better than either of the parents. A modified MS callus induction medium (CIM) with 1.5mg/l 2,4-D + 0.5mg/l Kn resulted impressively higher callusing response (30.2%) with nodular calli than SK3>MS>N6. High frequency (12.8%) of albino-free green plant regeneration with well developed rooting was achieved in RM medium (a modified MS) containing 2mg/l BAP and 0.5mg/l NAA. A total 129 doubled haploid (DH) plants (each with 12 pairs of chromosomes) were recovered which maintained normal growth, set seeds and resulted in true breeding DH lines. Interpretation: The high throughput regeneration system is amenable for doubled haploid production from indica crosses. Besides, the present doubled haploid stock can serve as an ideal mapping population and as such targeted for marker aided selection for early development of drought tolerant rice varieties.

English

Sorghum is a tropical grass used for various purposes including food, animal feed, and biofuels source, but its in vitro propagation still found an obstacle due to tissue browning. This study aims to propagate and regenerate callus in the Indonesian sorghum hybrid, named Numbu cultivar from seed and meristematic leaf whorl explants. The combined effect of plant growth regulators (PGRs), namely 2,4-D and proline as well as NAA (naphthalene acetic acid) and BAP (6-benzyl amino purine) was investigated on embryogenic callus induction and regeneration, respectively. The results showed that callus induction was significantly faster in the seed explants compared to leaf whorl. The optimal medium used to obtain a large induction in the seed explants was MS salt containing 3 mg L-1 2,4-D and 5000 mg L-1 proline, which gave a 68% yield. Furthermore, the best medium used to obtain a high plant regeneration frequency was the MS medium containing 0.1 mg L-1 NAA and 3 mg L-1 BAP, which regenerated 78% of the callus into plantlet. The addition of 560 mg L-1 proline to the combination medium increased doubled the regeneration into the planlet. Based on the results, PGRs combinations are required to improve embryogenic callus induction and regeneration into planlet in sorghum. © 2022 Friends Science Publishers

The influence of combinations of alien translocations on in vitro androgenesis in spring common wheat (Triticum aestivum L.)

Backgr ound.The basic approach to the production of new common wheat genotypes involving introgressive hybridization entails a long-term process. Doubled haploid production could accelerate it. However, this method is not widely used in breeding programs due to its main limitation: the genotype dependence. Due to genetic differences between wheat and related species, it was assumed that alien genetic materials are different in their capacity to affect androgenesis. The effect of alien translocations on androgenesis has been shown earlier. The aim of this study was to develop a set of DH wheat lines containing a wheat-alien translocation in the genome and study the effect of alien translocations on androgenesis of anther culture in such lines.Materials and methods. The plant material included: the spring wheat cultivar ‘Novosibirskaya 16’, line Velut 991 carrying wheat-alien translocations 1RS.1BL from rye and 5BS.5BL-5SL from Aegilops speltoides Tausch, and four hybrid F3 generation lines (10-7, 14-8, 15-8, 15-12) from their crossing, differing in the content of alien translocations.Results.It was shown that parameters of androgenesis such as the number of embryo-like structures per 100 anthers, the number of albino regenerants per 100 anthers, and the number of green regenerants per 100 anthers varied depending on the line. The best -responding lines Velut 991, 10-7 and 14-8 are characterized by the presence of a 1RS.1BL wheat-rye translocation chromosome. Regeneration frequency of green plants was recorded to be 8,6%, 3,6% and 10,1% respectively. The values of the parameters for lines 15-12 (carrying 5BS.5BL-5SL translocation) and 15-8 (without translocations) did not differ significantly.Conclusion.Therefore, it can be concluded that the presence of the introgressive fragment of chromosome 5S did not affect the efficiency of androgenesis and the short shoulder of chromosome 1R carries genes that stimulated androgenesis in anther culture.

Multiple Plant Regeneration from Embryogenic Calli of Paulownia tomentosa (Thunb.) Steud.

The aim of this paper was to study the effect of plant growth regulators on callus induction and in vitro morphogenesis using various explants of Paulownia tomentosa to develop an efficient plant regeneration protocol. Different plant organ sections (leaves, apical shoot tips, petals, nodes, and internodes) were cultured as explants to identify the best in vitro explants responsive to callus induction and plant regeneration. Explants were cultivated on MS media supplemented with different concentrations of plant growth regulators (TDZ (Thidiazuron), BAP (6-Benzylaminopurine), kinetin, and NAA (1-Naphthaleneacetic acid). It was discovered that the addition of TDZ and NAA stimulated the induction of somatic embryogenesis. It was discovered that the MS medium with the combination of plant growth regulators BAP (35.5 µM) and NAA (5.4 µM) with the addition of 30.0 g/L maltose, 500.0 mg/L casein hydrolysate, and 250.0 mg/L L-proline was optimal for callus induction and multiple plant regeneration. The study of the regenerative capacity of various explants of Paulownia tomentosa in vitro showed that plant regeneration depends on the type of explant, and occurs in both ways, indirectly, through the formation of callus tissues and directly on the explant, without callus formation. As a result of this study, the efficient reproducible protocol of embryogenic callus formation and multiple shoot induction in vitro of Paulownia tomentosa was developed. This system provides a clear increase in the frequency of plant regeneration from 36.3 ± 3.4% to 38.6 ± 2.3% per embryogenic callus from leaves and apical shoot tips, respectively.

Improvement of plant regeneration efficiency using polyamines for double haploid production in wheat (Triticum aestivum)

Polyamines are low molecular weight nitrogen containing compounds present in all living organisms. These are considered to be a new class of plant hormones, and play a positive role in plant growth and developmental processes including cell division, in vitro organogenesis and somatic, and zygotic embryogenesis. In our study, three polyamines PUT, SPD and SPM were used alone and in combinations in plant regeneration medium for in vitro plant regeneration from embryo developed through wheat × maize cross for double haploid production in wheat. Usually, these embryos have low regeneration efficiency but polyamines addition alone and in combinations in plant regeneration medium enhanced plant regeneration frequency as compared to control. Lower concentration of polyamines (1 and 2 ppm) has been found to be effective as compared to higher concentration (5 ppm). The polyamine concentration 1 ppm found to be promising concentration to improve plant regeneration. The combination of polyamines PUT+SPD (0.5 ppm+0.5ppm) proved better than other combinations to enhance plant regeneration frequency. The combination of PUT+SPD+SPM (0.5 ppm + 0.25 ppm + 0.25 ppm) although reduced plant regeneration frequency but plants were more vigorous and healthy.

Transient expression of a TaGRF4-TaGIF1 complex stimulates wheat regeneration and improves genome editing.

Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1 (TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex (mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of mTaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, mTaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.

Plant regeneration and transformation of Trachyspermum ammi using Agrobacterium tumefaciens and zygotic embryos

BackgroundTrachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg’s B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. ResultsAccording to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. ConclusionsIn this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25–35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques.

Effect of Forchlorfenuron on Somatic Embryo Proliferation and Plantlet Regeneration in Oil Palm SUP-PSU1

In vitro propagation of palm oil is still an obstacle for mass propagation due to the low plant regeneration frequency. The objective of this study was to investigate the effect of N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) on a somatic embryo (SE) proliferation and plant regeneration of oil palm SUP-PSU1. At an approximate size of 5 mm, SE from chopping with a sharp razor blade at 100 times was cultured on oil palm culture medium (OPCM) with 0.1 mg/L dicamba and different concentrations CPPU. The results showed that 0.3 mg/L CPPU gave the highest SE proliferation at 100 % and the number of SEs at 5.16 embryos/tube after 4 weeks of culture. SE was transferred to Murashige and Skoog (MS) medium supplemented with 0.2M sorbitol, a secondary somatic embryo (SSE), which was induced at 66.67 % with 21.2 embryos/tube after 8 weeks. Furthermore, cultured SSE on PGR-free MS medium for 8 weeks gave the highest germination rate at 65 %, with the number of shoots at 2.29 shoots. Thus, this study provides new information for improving the plantlet regeneration system through somatic embryogenesis in oil palm.

In vitro plant regeneration from mature embryos of amphidiploid spelt Triticum spelta L.

The present study reports an efficient in vitro plant regeneration system for amphidiploid (2n = 42) spelt wheat (Triticum spelta L.). Plant regeneration from mature embryos of winter spelt variety “Europe” was carried out as a two-step process. The first step was callus induction on a medium supplemented with 2 mg L−1 dichlorophenoxyacetic acid and 10 mg L−1 silver nitrate resulting in 96% callus formation. The second step resulted in 30% plant regeneration frequency from calluses transferred to Murashige and Skoog medium supplemented with 2 mg L−1 6-benzylaminopurine, 0.5 mg L−1 1-naphthaleneacetic acid, and 10 mg L−1 silver nitrate. Regeneration medium supplemented with 1 mg L−1 6-benzylaminopurine, 0.2 mg L−1 picloram, and the hormone-free medium turned out to be less effective in our experiments. Regenerated plants formed roots after transfer to half-strength Murashige and Skoog medium supplemented with 0.7 mg L−1 indole-3-butyric acid. About one-third of the resulting regenerated plants that formed roots were transferred to soil. The inter-simple sequence repeat markers were used to confirm the genetic homogeneity of regenerated plants. Thus, our regeneration protocol can be used for in vitro regeneration of spelt plants from mature embryos with minimal risk of genetic variability and provides an essential step towards developing an efficient strategy for spelt genetic transformation.

Grain barley starch as a gelling agent of nutrient media for cultivation of anthers and isolated embryos of H. vulgare L.

Aim. Determination the possibility of agar replacement with normal and waxy type starches produced from barley seeds in the nutrient media for spring barley haploid production. Methods. Anthers of spring barley line DH00-126 were inoculated on inductive media containing N6 macro-, MS micronutrients, organic supplements, maltose (9.0 %) and differed in solidifying agents (agar, starch from barley seeds or chemically modified starch D5a-1). Trophic capacities of barley starch were determined both in anther and embryo culture of barley. Results. It was determined that barley starch of waxy type was not suitable for solidifying of nutrient medium. Use of normal type barley starch (6.5 %) resulted in sufficient decrease in the frequency of green plant regeneration. At the same time, this modification had no effect on the number of morphogenic anthers. Barley starch was inferior to pea starch in the ability to support seedling growth in embryo culture in vitro. Conclusions. Barley starch of normal type has worse trophic capacities in comparison to pea starch in embryo culture. It was appeared barley starch to be a less suitable gelling agent of medium for spring barley haploid production in anther culture in vitro than agar and chemically modified starch D5a-1.
 Keywords: Hordeum vulgare L., anther culture in vitro, embryo culture in vitro, agar, barley starch.

ИЗМЕНЧИВОСТЬ И НАСЛЕДУЕМОСТЬ РЯДА ПРИЗНАКОВ У ПРОСТЫХ ГИБРИДОВ F1 КУКУРУЗЫ ПРИ ПОНИЖЕННОЙ ТЕМПЕРАТУРЕ И В ОБЫЧНЫХ УСЛОВИЯХ

The values of heritability coefficients of parental genotypes were determined for such characters as "frequency of callus formation", "frequency of somatic embryogenesis","frequency of plant regeneration" under normal and stressful in vitro conditions in simple maize F1 hybrids. The highest value of the coefficient of heritability of maternal genotypes in traits studied in vitro was found for “frequency of somatic embryogenesis” at low temperatures. The variability and heritability of characters during the initial stages of plant development under the actionof low temperatures, as well as quantitative characteristics of the plant and male gametophyte, were evaluated. Hybrids in which the inbred lines A239 and 092 were used as maternal genotypes showed under stress higher indices of the studied traits both invitro conditions and at the seed level. For the characters “number of grains per row” and “plant height”, the highest heritability coefficients of the interaction of maternal and paternal genotypes were noted. Hybrids A239xP502 and Co125xMK159 were selected by the higher values of the studied quantitative traits. Hybrid combinations with significantly high meanings of the most of the studied characters can be used to create cold-resistant and productive corn hybrids

Effects of Gibberellin and Abscisic Acid on Asexual Reproduction from Graptopetalum paraguayense Leaves

Graptopetalum paraguayense E. Walther is an edible succulent plant that is native to Mexico. When leaves of G. paraguayense are detached from the mother plant, shoots and roots regenerate from the leaf base. To investigate the endogenous hormonal regulation of plant regeneration in G. paraguayense, we evaluated the effects of applying gibberellin (GA), abscisic acid (ABA), and phytohormone biosynthesis inhibitors to leaves detached from the mother plant. GA3 and paclobutrazol did not affect plant regeneration of G. paraguayense, whereas ABA treatment significantly suppressed plant regeneration. When leaves were treated with the ABA biosynthesis inhibitor fluridone at 10 μM, the frequency of shoot regeneration was more than twice that of the control on the 6th day after leaf detachment. In addition, when both fluridone and ABA were applied at the same time, the frequency of plant regeneration decreased to control levels or below. Endogenous ABA in leaves was approximately 1.3 ng gFW−1 just after leaf detachment, decreased to 0.3 ng gFW−1 by the 1st day after leaf detachment, and increased to 3.0 ng gFW−1 by the 9th day after leaf detachment. These findings indicate that ABA plays an important role in suppression of plant regeneration in G. paraguayense. Although plant regeneration during storage and transportation currently reduces the economic value of G. paraguayense, our findings may provide a method to delay unwanted plant formation at the leaf base.

Morphological and histological analysis of anther-derived embryos of coconut (Cocos nucifera L.)

Doubled-haploids are a great source of material for heterozygous coconuts to shorten the time taken for crop improvement through hybridization programs. However, low frequency of embryo conversion and formation of weak plantlets are the major limitations in the protocol. In the present study anther-derived embryos were analysed at cellular level through the histological observations to understand the occurrence of shoot differentiation in those embryos. Attempts were also made to optimise the 6-Benzylaminopurine (BAP) concentration in the regeneration medium. Among the tested BAP levels (5, 15, 25, 35, 45, 55 μM), the regeneration medium containing 25 and 35 μM gave rise to 51.5% sprouted embryos against the control that gave only 22.7%. Among the analysed anther-derived embryos sequential events of differentiation including the formation of provascular strands followed by vascular bundle, differentiation in to the growing point, giving rise to the secondary embryos and polarization in to the shoot and root pole were identified. The blunt embryos devoid any morphological sign of sprouting showed different cellular arrangement viz. haustorial structure without any meristematic point (47%), bipolar with shoot and root meristems (8%) and unipolar with either of the pole (45%). Thick haustorial cover lead to the physical dormancy inhibiting further development of the polar structures. The germinating embryos containing a single shoot gave rise to the healthy plants where as double, multiple or fused shoots formed the week plantlets. The findings can be used for further optimisation of the protocol to achieve a greater plant regeneration frequency. 1. Culture media supplemented with increased levels of 6-Benzylaminopurine (BAP) enhanced the regeneration frequency of anther-derived embryos. 2. Thick haustorial tissue created an obstacle effect on further development of the differentiated shoot apical meristem. 3. Formation of clustered plants through secondary embryogenesis or shoot multiplication at the early stage of plant regeneration caused to give rise to the week plants.

Effect of the plant growth stimulant zeatin on regeneration capacity of some Physalis species in vitro culture

The aim of the study was to find an efficient culture medium for regeneration of Physalis species in vitro to provide their further propagation ex vitro and obtain fructiferous plants from the regenerants. Physalis peruviana L., P. ixocarpa Broth. (cv. Likhtaryk), and P. pubescens L. (cv. Zarynka) were taken as plant material for the research. Plant introduction into culture and regenerant production were carried out in vitro; the rooting of mature plants and obtaining plants with ripe fruits took place in a greenhouse and in open ground (ex vitro). To obtain regenerants, we used Murashige and Skoog (MC30) medium supplemented with the growth stimulant zeatin (Zea) at a concentration of 0.5–3 mg/l. The growth stimulant 6-benzylaminopurine (BAP) was used to elongate the regenerant stalks, and the growth stimulator α-naphthylacetic acid (NAA) was used to initiate root formation. Plant regeneration frequency and the number of regenerants per explant served as indicators of the efficiency of various zeatin concentrations on the physalis regenerative capacity. The most effective media for the shoot regeneration from cotyledonous leaf explants were MC30 + 1 mg/l Zea and MC30 + 2 mg/l Zea. Regeneration frequency on these media was 46.15 % and 53.84 % for P. ixocarpa (cv. Likhtaryk), 38.46 % and 45 % for P. peruviana, and 27 % and 34 % for P. pubescens (cv. Zarynka) respectively. The emerged regenerants were separated from explants and transferred to MC30 medium supplemented with 1 mg/l of BAP + 0.1 mg/l of NAA for stalk growth and rooting. After a month of cultivation, juvenile plants were obtained. They were transferred to a greenhouse for adaptation, and later to open ground at the experimental plot. Three months after the regenerant emergence, we obtained fertile plants, which bloomed and bore fruit. The regenerants for domestic varieties of P. ixocarpa (cv. Likhtaryk) and P. pubescens (cv. Zarynka) were obtained for the first time. We established a direct relationship between the concentration of zeatin and both the frequency of plant regeneration and the number of regenerants per explant.

Callus Induction and Regeneration from Anther Cultures of Indonesian Indica Black Rice Cultivar

The assembly of superior varieties and collection of rice germplasm involves the process of selecting and storing elders that have superior genotypic properties and phenotypes. The anther culture techniques on indica black rice cultivar have a high difficulty factor to get plants, because of the low regeneration ability at the plant formation phase from the anther callus. This study aimed to investigate the influence of the cold-pretreatment time on anther, the combination of plant growth regulators (PGR’s) concentrations, and putrescine concentrations in media for the increase callus induction and plant regeneration of indica black rice. The optimization of the cold pre-treatment time was important to obtain the high-frequency callus induction, which showed that anther at the 4°C for 8 days formed the high callus induction (20%). To accelerate the callus induction, the application of 20 µM putrescine in the MS medium could produce more friable embryogenic callus for 24 days with 27% of callus formation. Generally, the optimal medium for the high frequency of callus induction contained 2 mgL−1 NAA+0.5 mgL−1 Kinetin+20 µM putrescine. Especially indica black rice cultivars, the best media to get a high plant regeneration frequency were N6 media containing the combination of 2 mgL−1 IAA and 2,5 mgL−1 Kinetin. The total callus regenerated to plantlet about 12.5%. The study of the callus induction and in-vitro plant regeneration medium for indica black rice were still important to develop to get the best result for other cultivars.