De novo shoot organogenesis: from art to science

In vitro shoot organogenesis and plant regeneration are crucial for both plant biotechnology and the fundamental study of plant biology. Although the importance of auxin and cytokinin has been known for more than six decades, the underlying molecular mechanisms of their function have only been revealed recently. Advances in identifying new Arabidopsis genes, implementing live-imaging tools and understanding cellular and molecular networks regulating de novo shoot organogenesis have helped to redefine the empirical models of shoot organogenesis and plant regeneration. Here, we review the functions and interactions of genes that control key steps in two distinct developmental processes: de novo shoot organogenesis and lateral root formation.     

Role of AUX1 in the control of organ identity during in vitro organogenesis and in mediating tissue specific auxin and cytokinin interaction in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. It has been widely accepted that auxin and cytokinin play an antagonistic role in the control of organ identities during organogenesis in vitro. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, we find that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced in both calli and specific tissues of young seedlings. Our results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The role of local biosynthesis of auxin and cytokinin in plant development

Plant hormones are tightly regulated in response to environmental and developmental signals. It has long been speculated that biosynthesis of hormones occurs broadly in plant organs and that intricate, spatiotemporal regulation of hormones in developing organ primordia is achieved through transport and signal perception. However, recent identification of genes crucial for biosynthesis of auxin and cytokinin reveals that localized hormone biosynthesis also plays an important role in organ growth and patterning.     

Isolation of <Emphasis Type="Italic">HAG1</Emphasis> and its regulation by plant hormones during in vitro floral organogenesis in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

Floral organs have been successfully induced from the regenerated floral buds of Hyacinthus orientalis L. by precisely controlling exogenous hormones in the medium. Under high concentrations of cytokinin and auxin, the regenerated floral bud produces only tepals. However, at reduced levels of the hormones, the regenerated floral bud can produce stamens and/or carpels with ovules. To understand the molecular mechanism of hormone-regulated flower development, a MADS-box gene, HAG1, which is homologous to AGAMOUS (AG) in Arabidopsis, was isolated from the floral tissues of Hyacinthus. Overexpression of HAG1 in Arabidopsis created flower phenotypes resembling those of the apetala2 mutant and AG transgenic Arabidopsis plants. Furthermore, the HAG1 expression pattern was similar to that of AG, confirming that HAG1 is the ortholog of AG in Hyacinthus. HAG1 mRNA was first detected in cultured explants at day 5 in the medium containing high levels of cytokinin and auxin, which could induce floral regeneration in vitro. However, no HAG1 mRNA was detected in the cultured explants until day 10 in media with low or no hormones. Further, HAG1 mRNA was detected in the stamens and carpels of regenerated floral buds, but not in the tepals. Our data support the hypothesis that hormone-regulated HAG1 activity is required for the induction of floral buds and the determination of floral organ types during the regeneration of floral buds.     

Arabidopsis mutants with increased organ regeneration in tissue culture are more competent to respond to hormonal signals

Shoots and roots can be regenerated through organogenesis in tissue culture by subjecting plant explants to the appropriate regime of hormone treatments. In an effort to understand the control of shoot organogenesis, we screened for mutants in Arabidopsis thaliana (L.) Heynh. Columbia ecotype for enhanced shoot development at sub-optimal concentrations of cytokinin. Mutants in four different complementation groups were identified, one of which represents a new locus named increased organ regeneration1 (ire1) and another that is allelic to the previously identified pom1/erh2 mutant. Although the mutants were selected for their response to cytokinin, they were neither hypersensitive to, nor were they over-producers of cytokinins. The mutations identified in this study not only promote more robust shoot production in tissue culture, but also enhance green-callus and root formation. We interpret this to mean that, in tissue culture, IRE genes act before organ specification during the time when root explants acquire the competency to respond to organ formation signals. In normal plant development, IRE genes may down-regulate the competency of vegetative tissue to respond to hormonal signals involved in shoot and root organogenesis.     

花叶千年木单个生殖器官和营养器官的体外直接再生的控制——单个器官直接再生的规律性

花叶千年木(Dracaena fragrans cv.massangeana Hort．)的各种单个器官(花被片、花芽、花序分枝、花序、成年态营养芽和幼态营养芽)在离体培养中被愈伤组织直接再生了。在这些单个器官的再生期间,一些规律性现象被观察到了。首先,单个再生器官种类的范围与分离外植体的器官在植物个体发育中被分化的时期有密切关系。从植株个体发育某个时期(时期A)分化的地上部分器官上分离的外植体能够分别再生下面这些地上部分器官：稍晚于时期A分化的器官,与时期A同期分化的器官和早于时期A分化的所有器官。其次,在这个范围内,究竟再生哪一种器官被再生取决于培养基中外源生长素的浓度。随着2,4-D浓度从0．005mg／L逐渐升高到0．5mg／L,单个再生器官的种类将按如下的次序变化：营养芽,花序,花序分枝,花芽,花被片。这些规律性现象将被用于诱导一个给定的被子植物地上部分器官的直接再生。     

花叶千年木单个生殖器官和营养器官的体外直接再生的控制--单个器官直接再生的规律性

花叶千年木(Dracaena fragrans cv.massangeana Hort.)的各种单个器官(花被片、花芽、花序分枝、花序、成年态营养芽和幼态营养芽)在离体培养中被愈伤组织直接再生了.在这些单个器官的再生期间,一些规律性现象被观察到了.首先,单个再生器官种类的范围与分离外植体的器官在植物个体发育中被分化的时期有密切关系.从植株个体发育某个时期(时期A)分化的地上部分器官上分离的外植体能够分别再生下面这些地上部分器官:稍晚于时期A分化的器官,与时期A同期分化的器官和早于时期A分化的所有器官.其次,在这个范围内,究竟再生哪一种器官被再生取决于培养基中外源生长素的浓度.随着2,4-D浓度从0.005 mg/L逐渐升高到0.5 mg/L,单个再生器官的种类将按如下的次序变化:营养芽,花序,花序分枝,花芽,花被片.这些规律性现象将被用于诱导一个给定的被子植物地上部分器官的直接再生.     

Ethylene and shoot regeneration: hookless1 modulates de novo shoot organogenesis in Arabidopsis thaliana

We have investigated the role of ethylene in shoot regeneration from cotyledon explants of Arabidopsis thaliana. We examined the ethylene sensitivity of five ecotypes representing both poor and prolific shoot regenerators and identified Dijon-G, a poor regenerator, as an ecotype with dramatically enhanced ethylene sensitivity. However, inhibiting ethylene action with silver nitrate generally reduced shoot organogenesis in ecotypes capable of regeneration. In ecotype Col-0, we found that ethylene-insensitive mutants (etr1-1, ein2-1, ein4, ein7) exhibited reduced shoot regeneration rates, whereas constitutive ethylene response mutants (ctr1-1, ctr1-12) increased the proportion of explants producing shoots. Our experiments with ethylene over-production mutants (eto1, eto2 and eto3) indicate that the ethylene biosynthesis inhibitor gene, ETO1, can act as an inhibitor of shoot regeneration. Pharmacological elevation of ethylene levels was also found to significantly increase the proportion of explants regenerating shoots. We determined that the hookless1 (hls1-1) mutant, a suppressor of the ethylene response phenotypes of ctr1 and eto1 mutants, is capable of dramatically enhancing shoot organogenesis. The effects of ACC and loss of HLS1 function on shoot organogenesis were found to be largely additive.     

Simple hormonal regulation of somatic embryogenesis and/or shoot organogenesis in caryopsis cultures of Pogonatherum paniceum (Poaceae)

Pogonatherum paniceum (Poaceae) is a perennial plant with good potential for eco-recovery and ornamental function. This study presents in vitro culture systems of simple hormonal regulation of somatic embryogenesis and shoot organogenesis from mature caryopses. Mature caryopses of P. paniceum were grown on Murashige and Skoog medium with 3% sucrose (w/v) and various concentrations or combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Morphological development was analyzed by light microscope after histological sectioning. Four types of callus were induced by different concentrations of 2,4-D. Type I callus was regenerated via somatic embryogenesis; type II callus failed to produce any regeneration; type III callus had both somatic embryogenesis and shoot organogenesis capacities; and type IV callus only displayed shoot organogenesis capacity. Regarding hormone combinations used in this study, NAA only induced type IV callus and BAP only induced direct multiple shoot formation. The combinations of 2,4-D and NAA induced type III callus. Several of the regeneration pathways were simply controlled by one or two kinds of plant hormones. The established systems will be helpful for further research on the developmental mechanism of switch between somatic embryogenesis and shoot organogenesis.     

Inflorescence morphology, heterochrony, and phytogeny in the mimosoid tribes ingeae and acacieae (Leguminosae: Mimosoideae)

In earlier work (Grimes, 1992) on inflorescence morphology in the mimosoid tribes Ingeae and Acacieae I proposed that differences in inflorescence morphology result from three properties: the organization of components of the inflorescence and their relative positions; the hierarchical arrangement of the axes of the inflorescence and the position they assume in total tree architecture; and the heterochronic development of components of the inflorescence. Further work shows that the first two properties are better stated in terms of heterochrony; namely, that the organization of components of the inflorescence differs due to differences in timing of the development of organ systems and that the hierarchy of axes likewise differs due to heterochronic changes. Neither de novo origin of organs or organ systems nor suppression or loss of organs or organ systems accounts for the diversity in form. Observed heterochronic differences in the inflorescence structure may be divided into three types: spatial differences in the relationship between the unit inflorescence and the subtending leaf (hysteranthy); differences in the time of formation and/or the duration of whole axes; and changes in development pathways, leading to shoot dimorphism. These heterochronies are used as characters in a cladistic analysis, and it is shown that although some are homoplasious, many provide synapomorphies of clades of exemplars representing genera in the Ingeae and Acacieae.     

Effect of insulin and glucagon on aldolase turnover in rat liver

The effects of exogenous and endogenous insulin and glucagon on aldolase turnover in rat liver and blood were studied. Some effects of these hormones on the biosynthesis and degradation of hepatic aldolase were specified. The rate of the "de novo" synthesis of aldolase was investigated in hepatocyte mitochondria and in blood plasma. The exogenous and endogenous hormones were shown to produce different effects on the biosynthesis and spontaneous degradation of rat liver aldolase.     

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N⁶-side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.     

Axioms and axes in leaf formation?

Formation of leaves and floral organs involves down-regulation of meristem-specific homeobox genes, and de novo expression of genes for organ identity, growth and patterning. Genes required for all these aspects of organ formation have been identified. The challenge now is to establish how they interact to direct organogenesis.     

WOX11 and 12 Are Involved in the First-Step Cell Fate Transition during de Novo Root Organogenesis in Arabidopsis

De novo organogenesis is a process through which wounded or detached plant tissues or organs regenerate adventitious roots and shoots. Plant hormones play key roles in de novo organogenesis, whereas the mechanism by which hormonal actions result in the first-step cell fate transition in the whole process is unknown. Using leaf explants of Arabidopsis thaliana, we show that the homeobox genes WUSCHEL RELATED HOMEOBOX11 (WOX11) and WOX12 are involved in de novo root organogenesis. WOX11 directly responds to a wounding-induced auxin maximum in and surrounding the procambium and acts redundantly with its homolog WOX12 to upregulate LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) and LBD29, resulting in the first-step cell fate transition from a leaf procambium or its nearby parenchyma cell to a root founder cell. In addition, our results suggest that de novo root organogenesis and callus formation share a similar mechanism at initiation.     

Genetic approach towards the identification of auxin-cytokinin crosstalk components involved in root development

Phytohormones are important plant growth regulators that control many developmental processes, such as cell division, cell differentiation, organogenesis and morphogenesis. They regulate a multitude of apparently unrelated physiological processes, often with overlapping roles, and they mutually modulate their effects. These features imply important synergistic and antagonistic interactions between the various plant hormones. Auxin and cytokinin are central hormones involved in the regulation of plant growth and development, including processes determining root architecture, such as root pole establishment during early embryogenesis, root meristem maintenance and lateral root organogenesis. Thus, to control root development both pathways put special demands on the mechanisms that balance their activities and mediate their interactions. Here, we summarize recent knowledge on the role of auxin and cytokinin in the regulation of root architecture with special focus on lateral root organogenesis, discuss the latest findings on the molecular mechanisms of their interactions, and present forward genetic screen as a tool to identify novel molecular components of the auxin and cytokinin crosstalk.