Regeneration Of Fertile Plants Research Articles

Temporal Control of Morphogenic Factor Expression Determines Efficacy in Enhancing Regeneration.

Background: Regeneration of fertile plants from tissue culture is a critical bottleneck in the application of new plant breeding technologies. Ectopic overexpression of morphogenic factors is a promising workaround for this hurdle. Methods: Conditional overexpression of WUS and ARF5Δ was used to study the effect of timing the overexpression of these morphogenic factors during shoot regeneration from root explants in Arabidopsis. In addition, their effect on auxin-signaling activation was examined by visualization and cytometric quantification of the DR5:GFP auxin-signaling reporter in roots and protoplasts, respectively. Results: The induced expression of both WUS and ARF5Δ led to an activation of auxin signaling in roots. Activation of auxin signaling by WUS and ARF5Δ was further quantified by transient transformation of protoplasts. Ectopic overexpression of both WUS and ARF5Δ enhanced regeneration efficiency, but only during the shoot-induction stage of regeneration and not during the callus-induction stage. Conclusions: The overexpression of WUS and ARF5Δ both lead to activation of auxin signaling. Expression during the shoot-induction stage is critical for the enhancement of shoot regeneration by WUS and ARF5Δ.

Emerging Genome Engineering Tools in Crop Research and Breeding.

Recent advances in genome engineering are revolutionizing crop research and plant breeding. The ability to make specific modifications to a plant's genetic material creates opportunities for rapid development of elite cultivars with desired traits. The plant genome can be altered in several ways, including targeted introduction of nucleotide changes, deleting DNA segments, introducing exogenous DNA fragments and epigenetic modifications. Targeted changes are mediated by sequence specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspersed short palindromic repeats)-Cas (CRISPR associated protein) systems. Recent advances in engineering chimeric Cas nucleases fused to base editing enzymes permit for even greater precision in base editing and control over gene expression. In addition to gene editing technologies, improvement in delivery systems of exogenous DNA into plant cells have increased the rate of successful gene editing events. Regeneration of fertile plants containing the desired edits remains challenging; however, manipulation of embryogenesis-related genes such as BABY BOOM (BBM) has been shown to facilitate regeneration through tissue culture, often a major hurdle in recalcitrant cultivars. Epigenome reprogramming for improved crop performance is another possibility for future breeders, with recent studies on MutS HOMOLOG 1 (MSH1) demonstrating epigenetic-dependent hybrid vigor in several crops. While these technologies offer plant breeders new tools in creating high yielding, better adapted crop varieties, constantly evolving government policy regarding the cultivation of plants containing transgenes may impede the widespread adoption of some of these techniques. This chapter summarizes advances in genome editing tools and discusses the future of these techniques for crop improvement.

In Vitro Plant Regeneration of Zenia Insignis Chun.

Zenia insignis Chun is a large, fast-growing deciduous tree. In this study, we successfully developed a reliable and efficient protocol for the regeneration of fertile plants via callus induction from leaf segments of young Z. insignis seedlings. The best results were obtained with a medium containing 11.00 μM 6-benzyladenine (6-BA), 1.20 μM indole-3-butytric acid (IBA), and 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), which yielded morphogenic callus within 2 weeks at a frequency of 62.23%. We tested the effect of IBA alone and in combination with 6-BA on the bud differentiation response of Z. insignis callus. Shoots differentiated normally when cultured on differentiation medium containing 6.00 μM 6-BA and 1.20 μM IBA. Regenerated buds elongated successfully in medium containing 1.20 μM gibberellic acid (GA3). The elongated shoots were then transferred to Murashige and Skoog basal medium supplemented with various combinations of naphthalene acetic acid (NAA) for root induction; well-developed roots were achieved on MS basal medium supplemented with 0.01 μM NAA at a rooting rate of 89.23%. Rooted plantlets were successfully acclimatised to a greenhouse at a survival rate exceeding 90.00%.

Genetic Transformation of Hordeum vulgare ssp. spontaneum for the Development of a Transposon-Based Insertional Mutagenesis System.

Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100% in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.

Agroecological impact of an in vitro biotechnology approach of embryo development and seed filling in legumes

Ongoing global climatic changes and growing demographic pressure have increased demand for agronomic resources and affected the agroecosystem by provoking a number of abiotic stresses that, added to biotic ones, result in physiological and metabolic disorders. Such stresses ultimately impact yield when it most needs to be improved, and understanding and resolving them is a major scientific and agronomic challenge of this century. However, many species are difficult to breed for stress resistance and improved yield for a number of reasons, ranging from a long life cycle (woody species), a reduced genetic background (most self-fertile, cleistogamous legumes) or conversely extensive heterozygosity resulting from an outbreeding nature, and also due to the mainly multigenic origin of such resistances. Biotechnology-based breeding would be an efficient alternative but, for recalcitrant crops, many attempts at in vitro regeneration met with varying degrees of success and often limited to a few genotypes, hampering exploitation of biotechnology approaches. To reduce the risk of undirected somaclonal variations amongst regenerants and transformants, it is better to produce them through somatic embryogenesis that recognises a single-cell origin but whose feasibility is also limited amongst species. There is also a need to fix the resulting genome once a novelty is obtained to ensure efficient heritability of improved traits acquired, which takes several generations in conventional breeding. Acceleration of generations through flowering and fruit set in vitro has been developed in various species including legumes. Haplo-diploidisation in vitro also offers a unique alternative to conventional methods, as it yields novel genetic combinations following doubling of haplotypes and regeneration of fertile plants having gained homozygosity within a single generation. This review will examine the relationships between embryogenesis, stress and its impact on in vitro development of novel genotypes more apt for a sustainable agriculture.

Fertile plant regeneration from cryopreserved calli of Oryza rufipogon Griff. and assessment of variation in the progeny of regenerated plants

A protocol was developed for preservation of calli of Oryza rufipogon Griff. in liquid nitrogen (-196°C). Optimal conditions for cryoprotection, pre-freezing and post-thaw recovery of calli were identified. Cryoprotectant treatment at low temperature, pre-freezing in isopropanol bath for 1h at -70°C, rapid thawing and proper removal of cryoprotectant were critical for post-thaw survival. Genetic fidelity of the R1 plants obtained from cryopreserved, cryoprotectant-treated and untreated calli was assessed by phenotypic and molecular characterization. Comparison of phenotypic characters with seed-derived control plants showed no significant variation in the agronomic characters, but seed physical characters showed significant reduction in all the in vitro generated plants. Molecular data generated using 26 rice simple sequence repeat markers showed 4.78-7.25% change from control. Results suggested that both callus induction and cryopreservation induced heritable variations in O. rufipogon. In addition, a combination of phenotypic and molecular characterization using an appropriate marker provided better insight into genetic fidelity of recovered plants.

Organogenesis of Phaseolus angularis L.: high efficiency of adventitious shoot regeneration from etiolated seedlings in the presence of N6-benzylaminopurine and thidiazuron

A step-wise procedure for the regeneration of fertile plants by organogenesis from cultures of the economically important Phaseolus angularis L., cultivars: KS-6, KS-7 and KS-8 using etiolated seedlings was established. Pre-culture of 5-day old seedling explants with MS (Murashige and Skoog (1962) Physiol Plant 15:473–493) + B5-vitamins (Gamborg et al. (1968) Exp Cell Res 50:151–158) liquid medium containing either 5.0 µM TDZ or 5.0 µM BAP under dark condition was essential for organogenesis. Bud growth and shoot multiplication were stimulated by reducing the BAP concentrations from 5.0 to 2.5 µM after 3 weeks. The maximum frequency of shoot induction was 65.2% (33.8 ± 2.54 shoots/explant) in cultivar KS-8 followed by KS-7 34.6% (23.4 ± 1.91 shoots/explant) and KS-6 30.6% (21.2 ± 2.28 shoots/explant). The multiplied buds elongated after transferring to solid MSB5 medium supplemented with 4.0 µM GA3, 12.5 µM AgNO3 and 0.4 µM IBA. Up to 98% rooting efficiency of was obtained when the shoots were pulse-treated with liquid medium containing 4.5 µM IBA for 10 min. The rooted plantlets were transferred to pots in the greenhouse, where they grew, mature, flowered and bared pod normally. The efficient shoot bud induction capability was found to be cultivar dependent. All the three cultivars tested formed multiple shoots. This efficient and rapid regeneration system may also be helpful for Agrobacterium- or particle gun-mediated transformation for this important legume crop.

Regeneration and transformation of an elite inbred line of maize (Zea mays L.), with a gene from Bacillus thuringiensis

Abstract Tissue culture conditions for regeneration of fertile plants from immature embryos were optimized using four local inbred lines of Zea mays . Immature embryos were cultured on five different media. The highest regeneration efficiency of 86.40% was observed for BR-6 at N6 modified media (supplemented with 1.0 mg L − 1 2,4-D, 25 mM L-Proline, and 100 mg L − 1 Casein Hydrolysate) while B-46, EX-285 and EX-295 gave a regeneration percentage of 57%, 77%, and 63%, respectively. A total number of 1646 immature embryos were bombarded by a gene gun using tungsten micro projectiles coated with a multi-gene plasmid pCMAC, containing gusA (reporter gene), hpt (hygromycin phosphotransferase), and cry 1Ac genes driven by maize ubiquitin promoter. Regenerates were selected on a medium containing 40 μg/L hygromycin and then screened with GUS assay. Thirty-five transgenic lines were obtained with a transformation frequency of 2.21%. The integration and expression of cry 1Ac was confirmed with PCR, Southern Blot analysis, ELISA, and Western Blot analysis. The studies confirmed that cry 1Ac gene has been stably transformed into the local inbred line (BR-6) and caused 80–100% mortality to armyworm ( Spodoptera frugiperda ) in insect feeding bioassay.

Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. and C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5-8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30-35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea-an endangered medicinal plant.

Improved regeneration efficiency of a pearl millet (Pennisetum glaucum [L.] R.Br.) breeding line

This study reports the establishment of a highly efficient, reliable in vitro tissue culture system for the regeneration of fertile plants from immature zygotic embryo explants of pearl millet. Eight pearl millet genotypes were assessed for their amenability to tissue culture. The tissue culture responses obtained were strongly genotype-dependent. One of the most regenerable genotypes, 842B, was selected for further screening. The influence of the ethylene inhibitor silver nitrate, the osmoprotectant L-proline, and high carbohydrate pulses were examined with respect to callus induction, production of embryogenic callus and somatic embryo regeneration to form shoots and roots. Supplementation of the callus induction medium with L-proline in combination with maltose produced the most significant improvement in the regeneration capacity of genotype 842B resulting in, on average, eighty regenerants per cultured immature zygotic embryo.

Regeneration of fertile plants from isolated tobacco zygotes byin vitro culture

Living zygotes of tobacco (Nicotiana Tabacum L.) SR-1 were isolated and culturedin vitro by the microculture technique. Fertile plants were regenerated from the calli derived from cultured zygotes via organogenesis. Ovules were collected 120 h after pollination and used as feeder. MS combined with KM8p was selected as basic medium in the experiment. Zygotes isolated from ovules 108 h after pollination turned out to be suitable material forin vitro culture. Over 80% such zygotes could divide and around 10% of them could grow into calli and regenerate fertile plants.

Regeneration of fertile plants from isolated tobacco zygotes by in vitro culture

Regeneration of fertile plants from isolated tobacco zygotes by in vitro culture

Prolific regeneration of fertile plants from green nodular callus induced from meristematic tissues in Lathyrus sativus L. (grass pea)

A new in vitro protocol was developed for prolific shoot regeneration from two elite genotypes of Lathyrus sativus L. Vegetative apical and axillary bud explants from greenhouse-grown plants were used to induce regeneration-competent green nodular callus. Callus was induced on two B5 basal media that contained thidiazuron in combination with α-naphthaleneacetic acid or indole-3-acetic acid. Although callus induction was achieved on both media, progression to the formation of green-nodular callus, a distinct morphological feature and prerequisite for regeneration, differed. Shoot elongation was achieved on medium that contained 6-benzylaminopurine, indole-3-acetic acid, and coconut water. Regenerated shoots were readily converted with high efficiency into sexually mature plants either by planting shoots that were induced for rooting on hormone-free B5 basal medium or by grafting of non-rooted shoots.

The role of biotechnologies in the development of sunflower cultures in the world

SUMMARY Sunflower improvement by conventional breeding is severely restricted by the availability of a rather limited gene pool owing to natural incompatibilities, even between related species, and by the time scale of most breeding programs. Therefore, much attention has been directed recently to the newly emerging and novel technologies of plant cell and molecular biology that provide a powerful means to supplement and complement the traditional methods of plant improvement. The concept of DNA-based markers has revolutionized our ability to directly access any part of the plant genome, and has led to new opportunities such as map-based cloning and directed plant breeding. Efficient regeneration of fertile plants from cultured cells and protoplats, combined with novel methods of DNA delivery and selection of transformed cells, has resulted in the production of transgenic sunflower. Agronomically useful genes, which confer resistance to insect pests and pathogens, have been introduced. However, further molecular improvement of sunflower could be limited most by the lack of our knowledge about, and access to, important and useful genes (e.g., those controlling multigenic traits like yield, and resistance to biotic and abiotic stresses). Therefore, high priority should be given to the development of ultra high density (UHD) linkage maps and the development of new tools for high-throughput genome and expression analyses.

Regeneration of Fertile Plants from Callus in Phaseolus polyanthus Greenman (Year Bean)

For the first time, plant regeneration of several domesticated genotypes of Phaseolus polyanthus Greenman (year bean) has been achieved. Thidiazuron in combination with indole-3-acetic acid was used to induce morphogenic, green nodular callus from explants that had been obtained either from greenhouse-grown plants or from in vitro -germinated seeds. Of the six genotypes of P. polyanthus tested, five produced shoots in vitro. Regenerated shoots that formed roots in vitro were established in the greenhouse, whereas non-rooted shoots could be established in vitro by grafting. Morphologically normal progeny plants were obtained from the greenhouse-established regenerants. However, by using the same procedure, no regeneration response was observed in two domesticated and two wild genotypes of P. coccineus L. (runner bean). This protocol should help achieve Agrobacterium - or particle bombardment-mediated genetic transformation to improve this important food legume.

Genetic transformation of wheat: progress during the 1990s into the Millennium

Wheat transformation technology has progressed rapidly during the past decade. Initially, procedures developed for protoplast isolation and culture, electroporation- and polyethylene glycol (PEG)-induced DNA transfer enabled foreign genes to be introduced into wheat cells. The development of biolistic (microprojectile) bombardment procedures led to a more efficient approach for direct gene transfer. More recently, Agrobacterium-mediated gene delivery procedures, initially developed for the transformation of rice, have also been used to generate transgenic wheat plants. This review summarises the considerable progress in wheat transformation achieved during the last decade.An increase in food production is essential in order to sustain the increasing world population. This could be achieved by the development of higher yielding varieties with improved nutritional quality and tolerance to biotic and abiotic stresses. Although conventional breeding will continue to play a major role in increasing crop yield, laboratory-based techniques, such as genetic transformation to introduce novel genes into crop plants, will be essential in complementing existing breeding technologies.A decade ago, cereals were considered recalcitrant to transformation. Since then, a significant research effort has been focused on cereals because of their agronomic status, leading to improved genetic transformation procedures (Bommineni and Jauhar 1997). Initially, the genetic transformation of cereals relied on the introduction of DNA into protoplasts and the subsequent production of callus from which fertile plants were regenerated. More recently, major advances have been accomplished in the regeneration of fertile plants from a range of source tissues, providing an essential foundation for the generation of transgenic plants. This review summarises procedures, vectors and target tissues used for transformation, high-lights the limitations of current approaches and discusses future trends. The citation of references is limited, where possible, to the most relevant or recent reports.

Factors influencing plant regeneration from protoplasts isolated from long-term cell suspension culture of recalcitrant Indica rice cultivar IR36

Factors influencing successful establishment of embryogenic cell-suspension cultures and plant regeneration from longterm cell suspension-derived protoplasts of the recalcitrant Indica rice cultivar IR36 were studied. The factors included cell and protoplast culture medium, protoplast culture procedure, the source of nurse cells, and the regeneration procedure. Embryogenic cell suspension cultures could only be established from mature seed-derived callus of IR36 in AA-based medium (Muller and Grafe, 1978). Protoplast-derived colonies could be obtained only using the filter-membrane nurse-culture procedure when Lolium multiflorum suspension cells served as nurse, rather than wild rice (Oryza ridleyi) and Japonica rice (Oryza sativa cv. Taipei 309) cells. The utilization of a two-step regeneration procedure led to regeneration of fertile plants from protoplasts isolated from 2-yr-old cell suspensions of IR36, one of the most important but recalcitrant rice cultivars.

Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa).

A simple mechanical method has been developed which allows the routine isolation of unfertilized and fertilized egg cells from ovules of Japonica and Indica rice varieties. In the experiments described, the majority of the egg cells and zygotes survived the isolation procedure when the donor plants were in a vigorous state. About 40% of the surviving zygotes underwent sustained development when cultured in Millicell inserts with a non-morphogenic rice feeder-cell culture. Nearly all zygote-derived callus cultures regenerated multiple shoots, which could be subsequently rooted with high efficiency. Zygote-derived plantlets matured to fertile plants when transplanted to soil. So far, about 80 independent plants each from the Japonica variety 'Taipei309' and the Indica variety 'IR58' have been regenerated. The potential of this single-cell regeneration system for marker gene-free transformation is discussed.

Establishment of a highly efficient regeneration system from leaf base segments of oat ( Avena sativa L.)

A reliable and efficient protocol for the regeneration of fertile plants derived from leaf base segments of young in-vitro-grown oat seedlings has been developed successfully. Callus induction and shoot regeneration were achieved when the basal region of young seedlings was cultured on auxin-containing medium. Callus induction efficiencies as well as regeneration frequencies were correlated with the developmental stage and the genotype of the explants. In five different genotypes of oat, an average of 25 plants per explant could be produced and for the most responsive genotype more than 50 regenerants per explant could be regenerated reproducibly. This high regeneration potential makes oat leaf bases a very attractive target for transformation.

Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties

The effects were studied of various carbohydrates and osmotic stress, created by high agarose or carbohydrate concentrations, on the regeneration of fertile plants from protoplast-derived colonies of several indies (IR43, Jaya, Pusa Basmati 1) and japonica (Taipei 309) rice varieties. Observations of the cultures developed on media containing one of these carbohydrates (cellobiose, fructose, glucose, lactose, maltose, mannitol, sorbitol or sucrose), each at 88 mM, indicated that maltose was the preferential carbon source for the proliferation of embryogenic callus and shoot regeneration. Maltose-containing medium induced shoot formation in 24-66% of the protoplast-derived tissues, depending upon the rice variety, compared to shoot regeneration from 4-32% of the tissues in sucrose-supplemented medium. Media containing 288 mM maltose or an equimolar combination of 88 mM maltose and 200 mM mannitol, caused water loss from calli and promoted the growth of embryogenic calli. These calli formed shoots with greater frequencies when subsequently transferred to shoot regeneration medium with 88 mM maltose. A medium containing 88 mM maltose and semi-solidified with 1.0% (w/v) instead of 0.5% (w/v) agarose had a similar beneficial effect on the growth of embryogenic calli and simultaneously supported high-frequency (48-55%) shoot formation. The optimum shoot regeneration frequencies (60-78%) were obtained when protoplast-derived colonies were serially cultured on to shoot regeneration medium containing 1.0% (w/v) agarose for 4 weeks, followed by a 2-week culture period on the same medium with 0.5% (w/v) agarose. Plants regenerated on medium containing maltose and/or 1.0% (w/v) agarose were phenotypically normal and fertile.