Mineral nutrition and plant morphogenesis

Summary Plant morphogenesis in vitro can be achieved via two pathways, somatic embryogenesis or organogenesis. Relationships between the culture medium and explant leading to morphogenesis are complex and, despite extensive study, remain poorly understood. Primarily the composition and ratio of plant growth regulators are manipulated to optimize the quality and numbers of embryos or organs initiated. However, many species and varieties do not respond to this classical approach and require further optimization by the variation of other chemical or physical factors. Mineral nutrients form a significant component of culture media but are often overlooked as possible morphogenic elicitors. The combination of minerals for a particular plant species and developmental pathway are usually determined by the empirical manipulation of one or a combination of existing published formulations. Often only one medium type is used for the duration of culture even though this formulation may not be optimal for the different stages of explant growth and development. Furthermore, mineral studies have often focused on growth rather than morphogenesis with very little known of the relationships between mineral uptake and morphogenesis. This article examines the present knowledge of the main effects that mineral nutrients have on plant morphogenesis in vitro. In particular, the dynamics of nitrogen, phosphorus, and calcium supply during development are discussed.     

Predicting optimal in vitro culture medium for <Emphasis Type="Italic">Pistacia vera</Emphasis> micropropagation using neural networks models

In this study, artificial intelligence techniques—specifically artificial neural networks (ANNs) in combination with fuzzy logic (neurofuzzy logic) or with genetic algorithms (ANNs–GA)—have been employed, as modeling tools, to get insight, to predict and to optimize the effect of several independent factors on four growth parameters during Pistacia vera micropropagation. Twenty-six media ingredients, including mineral ions (or salts), glycine, vitamins and plant growth regulators (PGRs) at different concentrations, were used as inputs and four growth parameters: proliferation rate, shoot length, total and healthy fresh weight as outputs on the models. The IF-THEN rules from neurofuzzy logic models have allowed discovering the positive (BAP, nicotinic-acid and pyridoxine-HCl) and negative (NO₃ ^?, Mg²⁺, Ag⁺ and gluconate^?) effects on the growth parameters and the fundamental role of BAP over all of them. Also, ANNs–GA technology has permitted to estimate the best combination of media ingredients to simultaneously maximize the four parameters of growth: 4.4 new shoots per explant; 28.7 mm length; 1.1 and 0.53 g total and healthy fresh weight, respectively, minimizing physiological disorders. In our opinion, the information obtained in this study is extremely useful to improve the massive multiplication of pistachio plant, in particular, but also demonstrate the ability of artificial intelligence technology to design plant tissue culture media with predictable and tailorable characteristics.     

The role of activated charcoal in plant tissue culture

Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.     

Factors influencing somatic embryo maturation, high frequency germination and plantlet formation in Terminalia chebula Retz.

The factors influencing somatic embryo maturation, high frequency somatic embryo germination, and plantlet formation were studied in Terminalia chebula Retz. Maturation of somatic embryo were influenced by a number of factors such as in vitro culture passage, concentrations of sucrose, levels of abscisic acid (ABA), basal media and media additive combinations. Maximum frequency of somatic embryo maturation (57.22 ± 2.02), was obtained on MS medium supplemented with 50 g/l sucrose. Different factors such as strengths of MS nutrients, plant growth regulators, media additives and their combinations controlling somatic embryo germination and plantlet formation were studied. High frequency of germination and plantlet formation (58.80 ± 1.47) were achieved by subsequent subculture of mature somatic embryos on MS medium containing 30 g/l sucrose and 0.5 mg/l benzyladenine (BA). However, although duration of in vitro passage of the callus tissue was critical, contribution of the combinations of plant growth regulators and media additives showed nugatory effect on somatic embryo maturation and germination as evident from variable responses.     

Design of tissue culture media for efficient Prunus rootstock micropropagation using artificial intelligence models

Establishing optimized protocols for micropropagation of some economical plants, such as Prunus sp., is still one of the most important challenges for in vitro plant culture researchers. As an example, micropropagation of GF677 hybrid rootstocks (peach × almond) are extremely dependent on the medium ingredients and a large undesirable proportion of GF677 shoots need to be discarded as a result of hyperhydricity and chlorosis. In this study, an artificial intelligence technique—specifically neurofuzzy logic—has been employed, as a modeling tool, to increase knowledge on the effect of 8 ion macronutrients (NH₄ ⁺, NO₃ ^?, Ca²⁺, K⁺, Mg²⁺, SO₄ ^2?, PO₄ ^2? and Na⁺; as inputs) on three growth parameters (outputs): total number of shoots per explant, healthy number of shoots per explant, and their bud number. The model delivered new insights, by three sets of IF–THEN rules, pinpointing the key role of NO₃ ^? and their interactions (NO₃ ^? × Ca²⁺ and NO₃ ^? × Ca²⁺ × K⁺) on all growth parameters measured. All growth parameters showed a high correlation ratio between experimental and predicted values being 77.48, 91.78 and 90.78 for total shoots, healthy number and bud number, respectively. Regression coefficients higher than 77 % together with statistical significant ANOVA (p < 0.01) indicated good performance of neurofuzzy logic models. Moreover, The model also can be used for inferring the best combination of ion concentrations to obtain high quality GF677 micropropagated shoots. In conclusion, we assess the utility of neurofuzzy logic technology in modeling complex databases, identifying new complex interactions among macronutrients, and inferring new results and valuable knowledge, which can be applied to design new plant tissue culture media and improve plant micropropagation.     

SHOOT TIP CULTURE OF HELIANTHUS ANNUUS—FLOWERING AND DEVELOPMENT OF ADVENTITIOUS AND MULTIPLE SHOOTS

Shoot multiplication of Helianthus annuus was optimal from half shoot apices cultured on MS media with 0.1–1.0 mg/1 benzyl adenine or kinetin. Auxins inhibited multiplication and promoted callusing. Rooting was poor and was not promoted by auxins. Flowering of multiple shoots was observed after as little as 3 wk of culture. A number of plant growth regulators and environmental conditions had no effect on flowering which supports the determinate apex theory for sunflower (Habermann and Sekulow, 1972). Adventitious shoots were induced on leaves of the multiple shoots in some inbreds.     

Rapid Shoot Propagation from Micro-Cross Sections of Kiwifruit (<Emphasis Type="Italic">Actinidia deliciosa</Emphasis> cv. ‘Hayward’)

Kiwifruit shoots can be rapidly propagated through a micro-cross section (MCS) system we established here. Optimal culture conditions were determined for different expiant types, section sizes, and concentrations of inorganic salts and plant growth regulators. Rates of survival and callus formation were higher in half-strength MS salts than in full-strength MS media. Similar performance (i.e., survival and callus formation) was achieved with section sizes of either 800 μm or 1200 μm. Proliferation efficiency was greatest when expiants from stem tissue were cultured on 1/2 MS supplemented with 4.5x10^-3 μM 2,4-dichlo-rophenoxyacetic acid and 4.6x10^-1 μM zeatin. The number of shoots averaged 2.61 per expiant, representing an efficiency of 94%. RAPD analysis revealed that the regenerated plants from our MCS system were genetically stable. These results show that the culturing of micro-cross sections from stem tissue is a powerful method for kiwifruit propagation.     

Cytogenetic effect of plant tissue culture medium with certain growth substances onAllium sativum L. meristem root tip cells

The effect of plant tissue culture medium with different concentrations and combinations of growth regulators (kinetin, indol-3-ylacetic acid, 2,4-dichlorophenoxyacetic acid) was evaluated on mitosis ofAllium sativum meristem root tip cells. Different combinations of growth regulators at low concentrations had no effect on induction of mitotic aberrations or inhibition of mitotic activity. Inhibition of mitotic activity, a tendency to chromosome stickiness and clumping and a slight increase in the frequency of mitotic aberrations were observed at higher concentrations. It may be proposed that plant tissue culture media have no direct effect on induction of mitotic aberrations in plant tissue culturesin vitro.     

Media derived from brown seaweeds <Emphasis Type="Italic">Cystoseira myriophylloides</Emphasis> and <Emphasis Type="Italic">Fucus spiralis</Emphasis> for in vitro plant tissue culture

In the present study, the effect of seaweed extract (SE) from Fucus spiralis (Fs), Cystoseira myriophylloides (Cm) and Laminaria digitata (Ld) on in vitro plant tissue culture was examined. Combination of 25?% of SE from Cm with 25?% of MS medium increased adventitious shoot regeneration from Nicotiana benthamiana leaf discs explants by 620?%, when compared to the conventional regeneration medium. Similarly SE from Fs and Ld enhanced regeneration by about 500?%. However, when increasing SE to 50?%, only Cm significantly enhanced shoot regeneration. The effect of SE was also evaluated on in vitro micropropagation of N. benthamiana, grape, plum and apricot by assessing shoot length, number of leaves and internodes. When used alone but at lower concentrations (2.5 and 12.5?%), SE from Fs and Cm resulted in at least the same efficacy as MS alone for micropropagation of N. benthamiana shoots. However, for micropropagation of grapevine, plum and apricot woody plants, a combination of 50?% of SE from Cm or Fs with 50?% of their conventional micropropagation media was necessary. Rooting was also enhanced in N. benthamiana and grapevine, and was correlated with their higher concentrations of indole acetic acid when compared to SE from Ld. This finding, in addition to mineral analysis data, suggests that SE of Fs and Cm contain necessary nutrients and growth regulators to allow their use as medium for in vitro plant culture.     

Dissecting the effects of nitrate, sucrose and osmotic potential on Arabidopsis root and shoot system growth in laboratory assays

Studying the specific effects of water and nutrients on plant development is difficult because changes in a single component can often trigger multiple response pathways. Such confounding issues are prevalent in commonly used laboratory assays. For example, increasing the nitrate concentration in growth media alters both nitrate availability and osmotic potential. In addition, it was recently shown that a change in the osmotic potential of media alters the plant's ability to take up other nutrients such as sucrose. It can also be difficult to identify the initial target tissue of a particular environmental cue because there are correlated changes in development of many organs. These growth changes may be coordinately regulated, or changes in development of one organ may trigger changes in development of another organ as a secondary effect. All these complexities make analyses of plant responses to environmental factors difficult to interpret. Here, we review the literature on the effects of nitrate, sucrose and water availability on root system growth and discuss the mechanisms underlying these effects. We then present experiments that examine the impact of nitrate, sucrose and water on root and shoot system growth in culture using an approach that holds all variables constant except the one under analysis. We found that while all three factors also alter root system size, changes in sucrose and osmotic potential also altered shoot system size. In contrast, we found that, when osmotic effects are controlled, nitrate specifically inhibits root system growth while having no effect on shoot system growth. This effectively decreases the root : shoot ratio. Alterations in root : shoot ratio have been widely observed in response to nitrogen starvation, where root growth is selectively increased, but the present results suggest that alterations in this ratio can be triggered across a wide spectrum of nitrate concentrations.     

Plant regeneration from cell suspensions of spinach

Friable calluses induced from root segments of spinach (Spinacia oleracea L.) with a high amount of growth regulators (indole-3-acetic acid 48.52 μM and gibberellic acid 10 μM) were suspended in liquid medium. The cell fraction sized between 100 and 200 μm was used to establish suspension cultures. Adventitious shoots and roots were obtained from the suspensions (3.2 x 10⁵ cells per ml) by procedures comprising successive subcultures on two or three different media. In both of these procedures, the composition of the second culture medium (concentrations of plant growth regulators) had a key influence on the organogenesis of the suspensions. Regenerated shoots elongated and rooted on different solid media. Plantlets transplanted in soil grew and developed normally until flowering and produced seeds. This revised version was published online in June 2006 with corrections to the Cover Date.     

Vegetative micro-cloning to sustain biodiversity of threatened Moringa species

Efficient vegetative cloning in vitro requires definition of plant growth regulator regimes for each genotype, and therefore formulation of a uniform culture protocol for a genetically heterogeneous wild or uncultivated plant population is often impossible. The likelihood of cloning a wide array of plant genotypes by avoiding the use of plant growth regulator(s) was explored with Moringa oleifera Lamk., Moringa stenopetala (Baker f.) Cufod, and Moringa peregrina Forssk. ex Fiori tree seedlings. Propagation was achieved by multiple shoot regeneration from the cotyledonary node of decapitated seedlings, followed by axillary shoot growth from single node shoot segments and rooting of excised shoots. All steps were accomplished on basal Murashige and Skoog medium without plant growth regulator supplements. The results revealed competence for generation of multiple shoots from cotyledonary node tissue, stimulated by repeated shoot harvest, in seedlings of all three tree species. Tens of plants per seedling were regenerated in about 4 mo from culture initiation. In a given species clone size was seedling-dependent, which presumably stems from genotypic variability among seedlings in regeneration ability in vitro. By this means the laborious search for a plant growth regulator regime suitable for organogenesis induction and adapted per genotype became redundant, and biodiversity of the seed germplasm could be maintained. The approach ideally suits establishment of clones of wild plants of endangered species, like those of the Moringaceae, species with high ability for producing supplementary shoots, and without the need to add plant growth regulators, including the rooting stage.     

Mesos components (CaCl2, MgSO4, KH2PO4) are critical for improving pear micropropagation

Pear accessions and species show a broad response to tissue culture media due to the wide genetic diversity that exists in the available pear germplasm. An initial study of mineral nutrition using a systematic response surface approach with five Murashige and Skoog medium mineral stock solutions indicated that the mesos factor (CaCl₂, MgSO₄, and KH₂PO₄) affected most plant responses and genotypes, suggesting that additional studies were needed to further optimize these three mesos components for a wide range of genotypes. Short stature, leaf spots, edge necrosis, and red or yellow coloration were the main symptoms of poor nutrition in shoot cultures of 10 diverse pear genotypes from six species. A surface response experimental design was used to model the optimal factor and factor levels for responses that included overall quality, leaf character, shoot multiplication, and shoot height. The growth morphology, shoot length, and multiplication of these pear shoots could be manipulated by adjusting the mesos components. The highest quality for the majority of genotypes, including five P. communis cultivars, P. koehnei, P. dimorphophylla, and P. pyrifolia ‘Sion Szu Mi’, required higher concentrations (>1.2× to 2.5×) of all the components than are present in Murashige and Skoog medium. ‘Capital’ (P. calleryana) required high CaCl₂ and MgSO₄ with low KH₂PO₄; for ‘Hang Pa Li’ (P. ussuriensis), low CaCl₂ and moderate to low MgSO₄ and KH₂PO₄ produced high-quality shoots. Suitable combinations of the meso nutrients produced both optimum shoot number and shoot length in addition to general good plant quality.     

Use of response surface methodology for optimization of a shoot regeneration protocol in <Emphasis Type="Italic">Basilicum polystachyon</Emphasis>

Response surface methodology (RSM) is a collection of techniques useful for analyzing and optimizing problems where several explanatory covariates influence a response. Although this technique is extensively used in various mixture experiments, its application in standardization of micropropagation protocols is limited. The theoretical developments of RSM are usually concerned with continuous data; hence, linear model theory becomes relevant. In plant tissue culture, in which the response variables are mostly numerical data, the development of RSM in a generalized linear model (GLM) setup is of interest from both a theoretical as well as an application perspective. In the present paper, RSM, as applicable for count data, has been used for modeling, analyzing, and optimizing in vitro regeneration of multiple shoots of Basilicum polystachyon, an important medicinal plant. The specific issues addressed herein are the determination of the optimum concentration of plant growth regulators (i.e., the range of variation in dosages of each covariate) at which the regeneration potential of shoot tip explants is expected to increase, selection of the appropriate growth function (response function) of shoot tip, and determination of the optimum levels of the explanatory variables (i.e., the different combination of dosages of various control factors) for experimentation. According to the present analysis, the optimum level combinations of growth regulators for regeneration of multiple shoots from shoot tip explants of B. polystachyon is 8.19 μM benzyladenine and 2.36 μM naphthalene acetic acid, with a response of approximately 12 regenerated shoots.     

Adventitious rooting of conifers: influence of biological factors

Vegetative propagation of superior conifer trees can be achieved, e.g., through rooted cuttings or rooted microshoots, the latter predominantly through in vitro tissue culture. Both techniques are used to achieve rapid multiplication of trees with favorable genetic combinations and to capture a large proportion of the genetic diversity in a single generation cycle. However, adventitious rooting of shoots (cuttings) is often not efficient due to various problems, such as scarcity of roots and cessation of their growth, both of which limit the application of vegetative propagation in some conifer species. Many factors are involved in the adventitious rooting of shoots, including physical and chemical ones, such as plant growth regulators, carbohydrates, light quality, temperature and rooting substrates, or media [reviewed by Ragonezi et al. (Trees 24(6):975–992, 2010)]. The focus of this review is on biological factors, such as inoculations with Agrobacterium rhizogenes, plant-growth promoting rhizobacteria and other endophytes, and mycorrhizal fungi, which were found to stimulate adventitious rooting. These microorganisms could contribute not only to adventitious root development but also to help in protecting conifer plants against pathogenic microorganisms, facilitate acclimation and transplanting, and contribute to more sustainable, chemical-free forests.     

Split-root system optimization based on the survival,growth and development of the model Poaceae Brachypodium distachyon

Split-root system has been developed to better understand plant response to environmental factors, by exposing two separate parts of a single root system to heterogeneous situations. Surprisingly, there is no study attempting to maximize plant survival, growth and root system structure through a statistically sound comparison of different experimental protocols. Here, we aim at optimizing split-root systems on the model plant for Poaceae and cereals Brachypodium distachyon in terms of plant survival, number of roots and their equal distribution between the two compartments. We tested the effect of hydroponic or soil as growing media, with or without change of media at the transplantation step. The partial or total cutting of roots and/or shoots was also tested in different treatments as it could have an influence on plant access to energy and water and consequently on survival, growth and root development. Growing plants in soil before and after transplantation in split-root system was the best condition to get the highest survival rate, number of coleoptile node axile roots and growth. Cutting the whole root system was the best option to have a high root biomass and length at the end of the experiment. However, cutting shoots was detrimental for plant growth, especially in terms of root biomass production. In well-watered conditions, a plant submitted to a transfer in a split-root system is thus mainly lacking energy to produce new roots thanks to photosynthesis or adaptive autophagy, not water or nutrients.     

Effects of wheat plant inoculation with cytokinin-producing microorganisms on plant growth at increasing level of mineral nutrition

Triticum durum Desf. plants were grown for 11 days in sand culture on nutrient solutions with optimum or lowered content of mineral nutrients. Thereafter, the level of mineral nutrition was increased in some portion of deficient plants. Two days before, plants were inoculated with cytokinin-producing microorganisms of the Bacillus genus. Nutrition deficiency resulted in a decrease in the rate of plant biomass accumulation, which was correlated with the level of active cytokinins in both roots and shoots. After improving the mineral nutrition of noninoculated plants, the rate of their biomass accumulation increased and, by the end of experiment, their shoot fresh weight was 1.5-fold higher than in deficient plants; however, it was still by 20% lower than in plants continuously growing at optimum mineral nutrition. Inoculation resulted in the considerable increase in the cytokinin content in shoots as compared with all other treatments. In this case, after the improvement of plant mineral nutrition, the rates of growth and relative biomass accumulation increased sharply; as a result, these plants had the highest dry and fresh weights. Thus, inoculation with cytokinin-producing bacteria was beneficial for plant growth after their transfer from deficient to sufficient mineral nutrition.     

Seasonal acquisition of mineral nutrients by a chalcid gall on lowbush blueberry

Seasonal accumulation of various mineral nutrients by galls of the chalcidHemadas nubilipennis on lowbush blueberry were examined in various phases of gall development. Levels of copper, nickel iron and zinc were higher in tissues of galls in initiation phase compared to tips of ungalled shoots and in general were higher than galls in growth and maturation phases. Levels of manganese and magnesium in gall tissues decrease throughout the season compared to ungalled shoots. Levels of calcium are less in gall tissue in initiation phase compared to ungalled shoots, and elevated in maturation phase. Patterns of mineral concentration within galls at various phases of development are related to known physiological roles of minerals in plant nutrition; however we conclude that patterns are variable and their significance in gall physiology requires further study.     

Somatic embryogenesis in Pinus halepensis Mill.: an important ecological species from the Mediterranean forest

Pinus halepensis Mill. is a common forest species in the Mediterranean area and it is important for environmental conservation. This study established a method of regenerating Pinus halepensis Mill. through somatic embryogenesis. The effect of culture medium (mineral salts, nitrogen source and plant growth regulators), collection date and seed family on embryogenic tissue initiation and proliferation in Pinus halepensis was analysed during the first steps of embryogenesis process. This study showed a marked effect of the culture medium tested as well as some significant differences among collection dates. Furthermore, the embryogenic tissue initiation was affected by the amino acid mixture in the culture medium and the proliferation stage was significantly affected by the combination of plant growth regulators. At the end of the maturation phase the presence of activated charcoal was also evaluated. Finally, maturation of embryogenic tissue was affected by the nitrogen source in the culture medium and these results were different for high and low mature embryo producing cell lines. To the best of our knowledge, this is the first report on Aleppo pine somatic embryogenesis describing a simple and efficient procedure for large-scale somatic embryo production.     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.