Advertisement
ASHS 2024 Annual Conference

 

Optimization of Tissue Culture Medium for Little-leaf Mockorange (Philadelphus microphyllus A. Gray) by Adjusting Cytokinin and Selected Mineral Components

Authors:
Razieh Khajehyar Department of Plant Sciences, University of Idaho, Moscow, ID 83844, USA

Search for other papers by Razieh Khajehyar in
This Site
Google Scholar
Close
,
Robert Tripepi Department of Plant Sciences, University of Idaho, Moscow, ID 83844, USA

Search for other papers by Robert Tripepi in
This Site
Google Scholar
Close
,
Stephen Love Department of Plant Sciences, University of Idaho, Moscow, ID 83844, USA

Search for other papers by Stephen Love in
This Site
Google Scholar
Close
, and
William J. Price Department of Mathematics and Statistical Sciences, University of Idaho, Moscow, ID 83844, USA

Search for other papers by William J. Price in
This Site
Google Scholar
Close

Abstract

Little-leaf mockorange is a native plant species with desirable characteristics for landscape use. The need to conserve specific genotypes and the difficulty of seed propagation and stem cutting propagation make axillary shoot micropropagation a good option for this species. A series of experiments were completed individually with the goal to improve in vitro propagation protocols by evaluating different types of cytokinins [benzylaminopurine (BA), kinetin (Kin), zeatin (Zea), meta-topolin (MT), and thidiazuron (TDZ)] at 0, 1.1, 2.2, 4.4, or 8.8 µM. Selected minerals (0 to 60 mM or 0 to 45 mM N, 0 to 500 µM or 0 to 100 µM Fe, 0 to 3 mM Ca, 0 to 1.5 mM Mg, or 0 to 1.25 mM P) were also tested separately in the tissue culture medium; the base medium was ½ strength MS in these mineral experiments. At the end of each experiment (8 or 12 weeks), plant growth characteristics including number of axillary shoots, shoot height, and dry weight were determined. Of the six cytokinins tested, Zea produced the largest increase in shoot growth. Supplementation with 1.1 µM Zea resulted in the most shoot dry weight, almost 2.5-fold more than control shoots. Shoots on 0.55, 1.1, or 2.2 µM Zea were at least 64% taller than control shoots. Shoots placed on regular ½ strength MS basal salts, described above, and/or media lacking the nutrient of interest, were used as a positive and negative control treatments. For each separate mineral tested, the best concentration for optimum shoot growth was the concentration of that mineral used in ½ strength MS medium. A medium containing mineral concentrations of 30 mM N, 50 µM Fe, 1.5 mM Ca, 0.75 mM Mg, and 0.625 mM P, and 1.1 µM Zea should be used to produce the optimum in vitro shoot growth of little-leaf mockorange.

Native plants have traits that improve sustainability, adaptability, drought tolerance, and biodiversity of urban landscapes when used in geographical regions representative of their natural habitat (Khajehyar and Tripepi 2020). Little-leaf mockorange (Philadelphus microphyllus A. Gray) is a species from the Hydrangeaceae (Wikipedia 2019). This species is a shrub native to the western United States (California, Colorado, Utah, Nevada, Wyoming, Arizona, Texas, and New Mexico) and grows in arid rocky slopes, cliffs, or pinyon-juniper to coniferous woods (Gardenia 2019; Lady Bird Johnson Wildflower Center 2015). Species within the mockorange genus have historically been propagated by seeds, summer soft-wood cuttings, hardwood cuttings, and layering (Dirr and Heuser 2006), but little-leaf mockorange can be difficult to propagate as ex vitro cuttings and fails to breed true from seed (Love S, University of Idaho, personal communication), meaning a more efficacious propagation system, such as micropropagation, would be advantageous.

Plant tissue culture, also known as in vitro culture, is the science of growing cells, organs, or tissues on a culture medium within a container after a plant part is isolated or separated from a parent plant. Plants are propagated and grown on a mineral-amended medium in the presence of other substances, such as plant growth regulators (PGRs), vitamins, and carbohydrates (George et al. 2007; Loyola-Vargas and Vázquez-Flota 2006). Approximately 20 different components interacting together create the optimum growth medium for most plant tissue–derived explants (Trigiano and Gray 2010).

Tissue culture is a fast and highly reliable method for asexual plant propagation for research and is commercially used by the greenhouse and nursery industries for routine plant production. Finding the optimum culture medium and effective treatments to establish shoots followed by increasing the number of axillary shoots is important if tissue culture is to be feasible. In addition, finding the best treatments to induce shoots to form roots is quite important to complete the micropropagation process (Khajehyar and Tripepi 2020). For efficient propagation of cultured species at each propagation stage, testing different basal salt formulations, different PGRs, or changing mineral concentrations in the medium are often necessary. The medium ingredients may differ from species to species due to their different nutritional or PGR requirements.

Testing different salt concentrations and cytokinin concentrations should result in improved in vitro shoot growth. Nitrogen (N) is taken up in the form of nitrate (NO3) or ammonium (NH4+) and is an important component of amino acids, amides, nucleic acids, proteins, enzymes, vitamins, coenzymes, and PGRs. Phosphorus (P) is necessary for producing ATP, ADP, and AMP and is necessary for basic metabolic processes, such as photosynthesis, carbohydrate catabolism, and transferring energy within the plants. Potassium (K) plays some important roles in water and energy relationships. It is also linked with plant cold hardiness and increases frost tolerance through decreasing cell sap osmosis, regulates the supply of CO2 by controlling the stomata openings, and is involved in cell division. Calcium (Ca) is another macronutrient, necessary for structural phenomena such as cell division, cell elongation, and cell structure. Calcium also acts as a cofactor with some enzymes participating in ATP and phospholipid hydrolysis. Magnesium (Mg) has important roles as the main component of a chlorophyll molecule and is required by some enzymes involved in P transfer. Mg is also necessary for formation of carbohydrates, fats, and vitamins, while enhancing P uptake and transport within plants. Iron (Fe) is necessary for photosynthesis, metabolic process, and enzyme activation. Fe is a very important component of cytochrome and nonheme iron proteins. Fe has a catalyst role in the formation of chlorophyll. Fe helps with various reactions, such as respiration, photosynthesis and reduction of nitrates and sulfates (Khajehyar 2021).

Skoog and his colleagues discovered the first cytokinin (kinetin: Miller et al. 1955) and then studied the effects of kinetin on increasing the number of cell divisions of tobacco callus. Skoog and coworkers also recognized the importance of balancing the ratio between exogenous auxin and kinetin in the medium as these PGRs affected the morphogenesis of callus of cultured tobacco (Skoog and Miller 1957; Smith 2012; Trigiano and Gray 2010). Cytokinins have many physiological roles including promoting cell division, shoot initiation, and shoot growth [inducing lateral bud formation and growth (axillary budbreak)], adventitious shoot formation, delaying of leaf senescence, regulating nutrient allocation, antagonizing auxin responses in plants, and helping induce environmental and pathogenic responses by plants. Cytokinins also inhibit root formation (Khajehyar 2021). Scientists and propagators routinely look for combinations of PGRs that can enhance shoot proliferation of different plant species, as the mineral and plant growth regulator requirements of plants can differ from each other not only family by family, but even species by species within a genus. Some researchers have tested the seed composition of plant species to optimize the tissue culture medium of the same species. Najafian Ashrafi et al. (2009) used the walnut (Juglans regia L.) seed composition to optimize the shoot culture medium for that species.

The objective of this study was to improve in vitro growth of a horticulturally superior selected little-leaf mockorange plant to increase the number of plantlets rapidly available for eventual commercial production. In this study, we focused on adjusting concentrations of several components including inorganic minerals, such as N, P, Ca, Mg, and Fe, and cytokinins.

Materials and Methods

Plant material and culture condition

In 2019, stems from little-leaf mockorange derived from a selection held at the University of Idaho Aberdeen Research and Extension Center, Aberdeen, ID, USA, were acquired by the Plant Tissue Culture Laboratory in the Plant Sciences Department at University of Idaho, Moscow, ID, USA. The original plant of this accession was collected from the Goshutes Mountains, south of Wendover in Elko County, NV, in 2012 by Dr. Stephen Love. This selected little-leaf mockorange plant was chosen based on several key landscape traits. First, this plant had a very compact and dense habit, not necessarily dwarf but had a shortened height and dense branching compared with typical plants of this species. Second, the plant had leaves that displayed an attractive silverish color. Most plants of the species have indistinct medium green leaves. Last, the plant lacked a species-common tendency to produce random, long, leggy water sprouts. Taken altogether, traits expressed by this selection provided an aesthetically pleasing plant that would complement any landscape (Fig. 1). Another important characteristic of this plant is that it failed to produce true-to-type from seeds, meaning that to maintain these exceptional traits, vegetative propagation was essential (Love S, University of Idaho, personal communication).

Fig. 1.
Fig. 1.

The selected little-leaf mockorange from Aberdeen Research and Extension Center, University of Idaho, Aberdeen, ID, USA (picture taken by Dr. Stephen Love).

Citation: HortScience 59, 1; 10.21273/HORTSCI17440-23

Leaves were removed from stems, then stems were soaked in dilute soap solution (2 drops of Tween-20 per 100 ml) for 5 min and washed with tap water. Stems were cut into pieces containing two to three nodes each, dipped into 70% ethanol (v/v) for 30 s, followed by placement into a 10% bleach solution (v/v) for 20 min. Finally, stem pieces were rinsed in sterile distilled water three times. Each stem piece was cut at both ends to remove damaged tissue and put into individual culture tubes (2 × 15 cm) containing 10 mL of half-strength Murashige and Skoog salts (½ MS), plus 0.5 mg·L−1 thiamine-HCl, 0.25 mg⋅L−1 nicotinic acid, 0.25 mg⋅L−1 pyridoxine-HCl, 1 mg⋅L−1 glycine, and 0.05 g⋅L−1 myo-inositol, pH = 5.6 (Murashige and Skoog 1962) supplemented with 0.5 µM BA, 3% sucrose, and 7 g⋅L−1 agar (A-111, PhytoTech Laboratory, Lenexa, KS). Three percent sucrose and 0.7% agar were used in all maintenance and experimental culture media, and all media were autoclaved at 121°C and 15 psi (205 kPa) for 25 min.

Shoot cultures were maintained on culture medium for at least 6 months and were subcultured monthly to stabilize the shoot cultures. Every subculture cycle was completed by cutting the stems into several pieces of ∼1.5 cm in length and placing six stem explants onto 25 mL of ½MS in baby food jars (195 mL) used for culture maintenance. For all experiments, six explants and 25 mL of culture medium were used in each jar. Explants were incubated in a SG-30S germinator (Hoffman Manufacturing Inc., Albany, OR) at 25 ± 1 °C under a 16-h photoperiod (cool-white fluorescent lamps), with 38 μmol·m−2·s−1 photosynthetic photon flux. This process was completed every month to maintain and increase the number of shoot cultures to use for producing stem explants for later experiments. Stable shoot cultures were used in all subsequent experiments.

Plant growth regulator cytokinin experiments

In an experiment designed to optimize conditions for axillary shoot proliferation and growth of little-leaf mockorange shoot cultures, different concentrations of six cytokinins were tested individually. Stem explants (1.5 to 2 cm) were placed on ½ MS medium supplemented with different cytokinins including BA, Kin, Zea, MT, 2iP, or TDZ, each at concentrations of 0, 1.1, 2.2, 4.4, or 8.8 µM. All PGRs were added to the culture media before autoclaving. This concentration range was selected for this experiment based on previous results with other native species tested in our laboratory (unpublished data). Other components in the media were at their standard amounts in ½ MS medium salts, as well as 30 g⋅L−1 sucrose, and thiamine, nicotinic acid, pyridoxine, glycine, and myo-inositol at the concentrations described previously. The pH of the media was 5.6. Shoot explants were transferred onto the culture medium in baby food jars, and six explants were placed in each jar. Explants were subcultured onto the same medium every 4 weeks for 3 months (two subcultures).

During cytokinin experiments, explants grown on culture medium supplemented with Zea at concentrations between 1.1 and 2.2 µM were visibly growing better than other shoot cultures on media supplemented with other cytokinins. Hence, another experiment with only Zea in a narrower concentration range was completed to determine the best concentration suitable for increasing shoot proliferation of little-leaf mockorange. Explants were put onto ½ MS medium supplemented with Zea at concentrations of 0, 0.55, 1.1, 1.65, or 2.2 µM and kept on the medium for 4 weeks, subcultured and placed on the same treatment for 4 more weeks.

Minerals experiments

The standard basal MS salt consisted of 60 mM N, 3 mM Ca, 1.5 mM Mg, 1.25 mM P, and 100 µM Fe. The custom media tested in these series of experiments were all obtained from BioWorld Molecular Life Sciences, Dublin, OH, USA. Each custom medium lacked one of the minerals (N, Fe, Ca, Mg, or P). A separate stock solution of each of these minerals was then made to later combine with the custom ½ MS to obtain the concentrations needed in each mineral experiment.

N experiment.

As one of the critical macronutrients, nitrogen was the first mineral evaluated at different concentrations for its effects on shoot proliferation and explant growth. Nitrogen in the forms of NH4NO3 and KNO3 (at a ratio of 2:1) at concentrations of 0, 7.5, 15, 30, or 60 mM was added into ½ MS salts that lacked N. Shoots placed on regular ½ MS basal salts, described previously, were used as a positive control. By the end of this experiment, the highest N concentration had negative effects on the shoot cultures, severely inhibiting their growth. A second experiment was completed to help refine a recommendation with N at concentrations of 0, 22.5, 30, 37.5, or 45 mM to avoid deleterious effects.

Fe experiment.

As an essential and often limiting micronutrient, Fe was the second mineral tested in the culture medium. Five concentrations of Fe (0, 0.5, 5, 50, or 500 µM) were used to identify the best Fe concentration in the culture medium to increase shoot multiplication and growth of little-leaf mockorange. Half-strength MS custom medium lacking Fe was prepared, and the media were supplemented with the selected Fe concentrations. The Fe in the stock solution was in the form of FeSO4.7H2O and Na2EDTA was added to the solution. At the end of the experiment, all plants on the highest concentration of Fe died. Another experiment with a reduced range of Fe concentrations was therefore completed with 0, 25, 50, 75, or 100 µM Fe in ½ MS custom medium without iron.

Ca experiment.

The next experiment was conducted testing different concentrations of Ca in the form of CaCl2.2H2O at 0, 0.75, 1.5, 2.25, or 3 mM into ½ MS tissue culture medium that lacked Ca. One set of regular commercial ½ MS culture medium (as positive control) was also used to determine the best concentration of Ca promoting growth of little-leaf mockorange shoot cultures.

Mg experiment.

The next experiment tested Mg (MgSO4.7H2O) at concentrations of 0, 0.375, 0.75, 1.125, or 1.5 mM in ½ MS tissue culture medium that lacked Mg and one treatment of commercial ½ MS culture medium (as positive control) was also used to determine the best Mg concentration to improve shoot growth of little-leaf mockorange.

P experiment.

P (KH2PO4) at concentrations of 0, 0.312, 0.625, 0.937, or 1.25 mM into ½ MS tissue culture medium that lacked P (custom medium) and one treatment of commercial ½ MS culture medium (as positive control) was tested on shoot cultures of little-leaf mockorange.

Shoot harvest and data collection

After 3 months on culture medium (2 monthly subcultures) for the cytokinins experiment or 2 months on the culture medium (one subculture) for the Zea, N, Fe, Ca, Mg, or P experiments, shoots were harvested, and data were collected. The growth parameters evaluated were percentage of survival based on the proportion of dead shoots, number of axillary shoots formed, length of the longest shoot on each explant, the number of roots (if any) on each explant, and the length of the longest root and shoot dry weight (biomass) for each individual explant. The percentage survival was based on the number of the living explants producing new growth. To determine the biomass, samples were dried at 70 °C for 72 h and then weighed. After weighing shoots, samples were ground in a mortar to a fine powder and sent to a tissue analysis laboratory (Brookside Laboratories, Inc., New Bremen, OH, USA) to determine tissue mineral concentrations. The laboratory applied a combustion method using a Carlo Erba 1500 C/N analyzer to estimate total N content (method B2.20, Miller et al. 2013). For the rest of the minerals, they used nitric acid and hydrogen peroxide in a closed Teflon vessel and digested in a CEM Mars Microwave and analyzed on a Thermo 6500 Duo ICP (method B4.30, Miller et al. 2013).

Statistical analysis

The cytokinin experiments [with two factors, including six types of cytokinins, each at five concentrations and four replicate jars (6 × 5 × 4)] along with an additional Zea and mineral experiments [one-way analyses with the five concentrations and four replicate jars (5 × 4)] were randomized complete block designs. All the data were analyzed using SAS software version 9.4 (SAS Institute Inc. 2016). A generalized linear mixed model (Proc GLIMMIX) was used to assess treatments. This model included cytokinin type and concentration in the cytokinin experiment, and Zea (in the second Zea study) or mineral concentration in Zea and minerals experiments. Treatments were considered as fixed effects and blocks as random effects. The models assumed a normal distribution for dry weight and shoot length, and a Poisson distribution with a log link for number of axillary shoots (Stroup 2014). For detectable effects, pairwise comparisons of marginal means were used to assess treatment differences. Roots were rarely observed for any of the treatments; hence, root data were excluded from statistical analyses.

Results

Cytokinin experiment.

Concentration of the various cytokinins had detectable effects on shoot growth, such as number of axillary shoots (Fig. 2A), shoot length (Fig. 2B), and dry weight (Fig. 2C) of little-leaf mockorange shoots in culture. Only shoot dry weight was affected by an interaction between cytokinins and their concentrations (Table 1, Fig. 2C). Because of this interaction, the effects of concentration on shoot dry weights were evaluated separately for each cytokinin (Table 1).

Fig. 2.
Fig. 2.

Effects of different cytokinins [benzylaminopurine (BA), kinetin (Kin), zeatin (Zea), meta-topolin (MT), dimethylamino purine (2iP), and thidiazuron (TDZ)] on in vitro growth of little-leaf mockorange shoots averaged over their concentrations for number of axillary shoots (A) and shoot length (B) of in vitro shoots. The interaction between cytokinins (BA, Zea, and TDZ) selected for their strong, positive, and negative effects and their concentrations on dry weight of little-leaf mockorange shoot cultures (C). Data are means (n = 4), and error bars indicate ± 95% confidence limits.

Citation: HortScience 59, 1; 10.21273/HORTSCI17440-23

Table 1.

Main effects and the interactions between cytokinins and their concentrations on the number of axillary shoots, shoot length, and dry weight of little-leaf mockorange shoot cultures. P values indicate statistical significance.

Table 1.

MT concentration affected the mean number of axillary shoots (P = 0.02; Table 2). MT at 8.8 µM produced the most axillary shoots (4.3-fold more than control shoots), whereas MT at 1.1, 2.2, and 4.4 µM increased shoot number less than MT 8.8 µM but still more than the control shoots (Fig. 3A). MT also affected shoot length, with shoots on 1.1 and 2.2 µM MT growing two times taller than control shoots (Fig. 3B).

Table 2.

The effects of cytokinin type [benzylaminopurine (BA), kinetin (Kin), zeatin (Zea), meta-topolin (MT), dimethylamino purine (2iP), and thidiazuron (TDZ)] on the number of axillary shoots, shoot length, and dry weight of little-leaf mockorange shoot cultures. P values with an asterisk indicate statistical significance.

Table 2.
Fig. 3.
Fig. 3.

The effects of different concentrations of MT on the number of axillary shoots formed by little-leaf mockorange shoot cultures (A). Comparison between different concentrations of MT (B) and BA (C) on shoot length of little-leaf mockorange cultures. The effects of different concentrations of TDZ on shoot length of little-leaf mockorange shoot cultures (D). The effect of 0, 0.55, 1.1, 1.65, or 2.2 µM zeatin on shoot length (E) and dry weight (F) of little-leaf mockorange. Data were means (n = 4), and error bars indicate ± 95% confidence limits.

Citation: HortScience 59, 1; 10.21273/HORTSCI17440-23

BA affected shoot length and dry weight (Table 2). BA changed shoot dry weight, in that increasing the concentration of BA from 1.1 to 4.4 µM decreased shoot dry weight by 28% as determined by pairwise comparisons, although this average weight was greater than the control (P = 0.0475). In contrast, shoots on the medium without BA weighed 52% more than shoots on 8.8 µM BA (Table 3). Shoots grew the tallest on BA at 1.1 µM, followed by those on 2.2 µM BA, but concentrations of BA at 4.4 or 8.8 µM decreased shoot length to even shorter than the control explants, although the lengths were statistically similar (Fig. 3C).

Table 3.

The effects of cytokinins [benzylaminopurine (BA), kinetin (Kin), zeatin (Zea), meta-topolin (MT), dimethylamino purine (2iP), and thidiazuron (TDZ)] and their concentrations on mean shoot dry weights of little-leaf mockorange cultures. Six different cytokinins were tested at five concentrations on shoot explants. Data were means ± 95% confidence limits (n = 4). An interaction was observed that indicated the pattern of change over concentration differed by cytokinin type.

Table 3.

Kin or 2iP also affected only shoot dry weight (Table 2). Shoots on medium supplemented with 2.2 µM Kin or higher weighed at least 1.5-fold more than control shoots as determined by pairwise comparisons. Shoots on 4.4 or 8.8 µM Kin also weighed at least 49% or 59% more, respectively, than shoots grown on 1.1 µM Kin (Table 3). Mean shoot dry weights among all 2iP concentrations were similar, yet they produced at least 70% more dry weight than control shoots (Table 3).

Zea significantly increased only shoot dry weight of little-leaf mockorange (Table 2). Similar to 2iP, any Zea concentration resulted in shoots producing at least 2.2-fold more dry weight than control shoots (Table 3). Mean shoot dry weights among the four Zea concentrations tested were similar (Table 3).

TDZ failed to improve any of the growth parameters of little-leaf mockorange shoot cultures (Table 2). All explants placed on culture medium supplemented with any concentration of TDZ died. Shoots growing on TDZ at first showed very little growth, and eventually died, especially at high concentrations (Fig. 3D).

In the second Zea experiment, shoots placed on medium with 1.65 µM Zea produced the most biomass (dry weight), and those on 0.55 µM Zea or higher concentrations grew taller than control explants (Fig. 3F and E). Shoots on 0.55, 1.1, or 2.2 µM Zea were at least 64% taller than control shoots.

Minerals experiments.

In the first experiment testing different N concentrations in the culture medium, increasing N from 0 to 30 mM increased the number of axillary shoots by 6-fold. Increasing N from 30 (mean value 1.8) to 60 mM (1.0 mean value) caused a severe decrease in the shoot number. The number of axillary shoots formed on explants grown on 30 mM N or the positive control (½ MS medium), were similar (Fig. 4).

Fig. 4.
Fig. 4.

Effects of different N concentrations on number of axillary shoots. Data were means (n = 4), and error bars indicate ± 95% confidence limits. The ½ MS treatment (positive control) contained 30 mM N.

Citation: HortScience 59, 1; 10.21273/HORTSCI17440-23

Regarding shoot length and dry weight, the same trend was seen if applying N from 0 to 60 mM with shoots on 30 mM N being 66% taller and producing 8-fold more dry weight than shoots on medium without N (Fig. 5A). In contrast, shoots on the positive control medium grew 38% taller (Fig. 5A) and produced 84% more dry weight compared with shoots on 30 mM N (Fig. 6A).

Fig. 5.
Fig. 5.

Effects of different N concentrations (first experiment, A), Fe (first experiment, B; second experiment, C), Ca (D), Mg (E), and P (F) concentrations on shoot length of little-leaf mockorange in tissue culture. Data were means (n = 4), and error bars indicated ± 95% confidence limits. The ½ MS treatment (positive control) contained 30 mM N, 50 µM Fe, 1.5 mM Ca, 0.75 mM Mg, or 0.625 mM P in each individual mineral experiment.

Citation: HortScience 59, 1; 10.21273/HORTSCI17440-23

Fig. 6.
Fig. 6.

Effects of different N (first experiment, A; second experiment, B), Fe (first experiment, C; second experiment, D), Ca (E), and P (F) concentrations on dry weight of little-leaf mockorange in tissue culture. Data were means (n = 4), and error bars indicated ± 95% confidence limits. The ½ MS treatment (positive control) contained 30 mM N, 50 µM Fe, 1.5 mM Ca, 0.75 mM Mg, or 0.625 mM P in each individual mineral experiment.

Citation: HortScience 59, 1; 10.21273/HORTSCI17440-23

In the second N experiment, only shoot dry weight differed among the treatments. Dry weights of shoots cultured on 22.5 or 30 mM N were almost 12-fold higher than dry weights of control shoots (Fig. 6B). Although statistically similar, dry weights of shoots on 37.5 or 45 mM N were lower than weights of shoots on 22.5 or 30 mM N (Fig. 6B).

Only shoot length and dry weight were affected by Fe concentrations. Increasing Fe from 0 to 50 µM increased shoot length 43% (Fig. 5B) and doubled the dry weight (Fig. 6C). The percentage of shoot survival on medium supplemented with 500 µM Fe was zero, as this concentration killed all the explants, as indicated by drastic reduction in shoot dry weight (Fig. 6C).

In the second Fe concentration experiment, shoot length (P = 0.02) and dry weight (P = 0.005) were also significant. Shoots grown on 25 µM Fe or higher grew at least 75% taller (Fig. 5C) and produced 70% more dry weight (Fig. 6D) than shoots grown on control medium.

Shoot length and dry weight of explants grown on media with varied Ca concentrations were significantly different (Figs. 5D and 6E). Plants grown on 1.5 mM Ca were 2.5-fold taller than control shoots, but shoots heights between different Ca concentrations were statistically similar. Shoot length resulting from culture on ½ MS control explants were taller than shoots grown on other Ca concentrations. Shoots grown on 0.75 mM Ca or higher, as well as positive control (½ MS) explants produced the most shoot biomass compared with the negative control (lacked Ca) shoots.

Only shoot length was significantly affected by changes in Mg concentration (P < 0.0001). Shoots grown on 0.75 mM Mg were tallest, although were shorter than shoots grown on ½ MS control medium (Fig. 5E). From Mg at 0 to 0.75 mM shoot height tended to increase, although shoot heights of explants on medium with any level of Mg were similar.

Shoot length and dry weight were significantly affected by P concentration. Shoots on medium supplemented with 0.31 mM P or higher grew at least 2.8-fold taller (Fig. 5F) and produced 3.6-fold more shoot dry weight (Fig. 6F) compared with shoots on the negative control medium.

Discussion

In this study, overall shoot growth, as indicated by increasing shoot length and dry weight of little-leaf mockorange, was promoted best by a medium supplemented with Zea, followed closely by medium supplemented with MT. Shoots treated with 1.1 µM MT produced 28% less dry weight and were 11% shorter than those on medium supplemented with 1.1 µM Zea. Similar results were reported from other studies showing Zea either by itself or in combination with another cytokinin promoted shoot proliferation of various plant species, such as the combination of Zea and BA for micropropagation of olive (Olea europaea L. cv. Moraiolo: Ali et al. 2009). Zea promoted more shoot proliferation for dwarf raspberry (Rubus pubescens Raf.) shoot cultures compared with BA alone (Debnath 2004). Zea combined with TDZ increased shoot multiplication of lingonberry (Vaccinium vitis-idaea L.: Debnath 2005; Ostrolucká et al. 2004). In contrast, Zea failed to increase shoot growth of tissue-cultured phlox (Phlox kelseyi) shoots (Khajehyar, unpublished data). Overall, using Zea within a range of 0.55 to 2.2 µM promoted shoot growth (more biomass production and taller shoots), which should help growers to obtain more healthy propagules within a shorter period of time. Zea is the natural form of the cytokinin, and the positive responses of little-leaf mockorange to this cytokinin were probably due to this fact.

The results obtained from this study were in agreement with that of other researchers studying and reporting the positive effect of MT on shoot proliferation, such as the positive effect of MT on improving axillary shoot number of firechalice (Epilobium canum garrettii: Alosaimi and Tripepi 2018); the positive effect of 2 µM MT on shoot number, dry weight, and rooting percentage of sea oats (Uniola paniculata L.: Valero-Aracama et al. 2010); and induction effect of MT at 5 µM on the best rate of shoot multiplication by Aloe polyphylla, an endangered medicinal and ornamental aloe species (Bairu et al. 2007).

TDZ failed to promote shoot proliferation of little-leaf mockorange grown in vitro. TDZ in this experiment showed inhibitory or toxic effects on explant growth. Increasing TDZ concentration increased its negative effect on shoot length and dry weight. TDZ is a synthetic cytokinin-like PGR, first introduced in Germany as a cotton defoliant (Arndt et al. 1976; Dewir et al. 2018). This PGR is resistant to cytokinin oxidase, hence is very stable in culture medium (Dewir et al. 2018; Mok et al. 1982). Unlike Zea, TDZ exhibits very slow metabolism within plant tissues (Mok and Mok 1985). TDZ resistance to cytokinin oxidase may cause accumulation of purine cytokinins in plant tissues (Dewir et al. 2018; Hare et al. 1994; Horgan et al. 1988). Zhang et al. (2006) stated that treatment of plants with TDZ induced accumulation of ethylene in plant tissues due to the expression of stress-related genes. Signaling of stress-related genes to produce proline or abscisic acid also resulted from treatment with TDZ (Dewir et al. 2018; Jones et al. 2007; Murch et al. 1999). The inhibitory effects of TDZ on plant tissue culture is also species dependent, and these effects may be more prominent for some plant species, such as little-leaf mockorange.

Cytokinins play key roles in long-distance signaling, a control mechanism for N assimilation in plants (Rubio et al. 2009; Sakakibara et al. 2006). These qualities may explain the promotive effects of cytokinins on shoot proliferation of explants in vitro, as N is critical for vegetative growth. Increasing nitrate supply in the soil and through plants’ roots induces the expression of genes related to regulating nitrate and carbon metabolism in plants, which can be mimicked by applying cytokinin in the culture medium (Brenner et al. 2005; Rubio et al. 2009; Scheible et al. 2004). Applying or increasing the concentration of cytokinins can enhance N utilization, resulting in more budbreaks and shoot proliferation.

Selecting the best PGR for the micropropagation can be critical especially for growers considering the price of different PGRs and the purpose for using them. Growers need to consider all aspects of their micropropagation goals and choose the best PGR for economic production and to meet proliferation goals. For little-leaf mockorange, the best cytokinin to use for in vitro growth of shoot cultures was Zea. However, the high cost of Zea may be prohibitive for some propagators, a situation in which a compound, such as MT, may prove to be more economically feasible.

Researchers have reported positive effects of applying N in the culture medium for different crops. Application of N in tissue culture medium improved shoot multiplication and growth of micropropagated Indian gooseberry (Phyllanthus emblica also known as Emblica officinalis: Sen and Batra 2011). Sen and Batra (2011) also stated that the best responses were found for shoots grown on media including commercially standard concentrations of N, which the results with shoot cultures of little-leaf mockorange confirmed.

Experiments demonstrated that Ca, Fe, Mg, or P were all essential for shoot growth of little-leaf mockorange, yet some of them had stronger effects. Ca, Mg, and P supplementation at any level within the experimental parameters clearly increased shoot dry weight compared with negative control shoots. Shoots grown on Ca-supplemented media grew at least 1.9 times taller than those on the control treatment. Also, addition of Ca resulted in at least 1.5-fold more shoot dry weight compared with the negative control. Supplementation resulted in similar results to the positive control, showing that using the Ca concentration in regular ½ MS medium can be sufficient for little-leaf mockorange optimum growth. Addition of Mg provided significant improvements only to shoot length. Lee and Fossard (1977) and Ramage (1999) stated that P is an important mineral both for explant growth and morphogenesis (Ramage and Williams 2002). Shoot lengths and dry weights were significantly improved by P supplementation to culture media.

To our knowledge, this research is the first study on micropropagation of any mockorange species. Using ½ MS medium supplemented with Zea may enable micropropagation of other native mockorange species and selected mockorange cultivars grown in nurseries.

Conclusion

In this study, little-leaf mockorange grew best on ½ MS medium supplemented with Zea within a range of 0.55 to 2.2 µM. If the price of the cytokinin is a key factor in choosing a cytokinin source, then MT at 2.2 µM is suggested for use instead to increase economic feasibility. Regarding the adjustment and optimization of the minerals in the tissue culture medium, standard concentrations of N, Fe, Ca, Mg, and P in ½ MS medium promoted shoot growth of little-leaf mockorange. Considering these results, 1.1 µM Zea should be added to standard ½ MS medium for optimum shoot growth and efficient micropropagation of little-leaf mockorange.

Future research could involve using surface modeling (statistical) experiments to refine further the critical concentrations of the tested minerals and PGRs used in this research. In addition, this research may be useful for micropropagation of other native mockorange species and commercially important mockorange cultivars, enabling nursery growers to propagate desired genotypes quickly.

References Cited

  • Ali A, Ahmad T, Abbasi NA, Hafiz IA. 2009. Effect of different media and growth regulators on in vitro shoot proliferation of olive cultivar ‘Moraiolo’. Pak J Bot. 41:783795.

    • Search Google Scholar
    • Export Citation
  • Alosaimi AA, Tripepi RR. 2018. Micropropagation of Epilobium canum Garretti (firechalice) by axillary shoot culture. HortScience. 53:6266. https://doi.org/10.21273/HORTSCI12396-17.

    • Search Google Scholar
    • Export Citation
  • Arndt FR, Rusch R, Stillfried HV, Hanisch B, Martin WC. 1976. SN: 49537, a new defoliant (abstr). Plant Physiol. 57:99.

  • Bairu MW, Stirk WA, Dolezal K, Van Staden J. 2007. Optimizing the micropropagation protocol for the endangered Aloe polyphylla: Can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin? Plant Cell Tissue Organ Cult. 90:1523. https://doi.org/10.1007/s11240-007-9233-4.

    • Search Google Scholar
    • Export Citation
  • Brenner WG, Romanov GA, Köllmer I, Bürkle L, Schmülling T. 2005. Immediate-early and delayed cytokinin response genes of Arabidopsis thaliana identified by genome-wide expression profiling reveal novel cytokinin-sensitive processes and suggest cytokinin action through transcriptional cascades. Plant J. 44:314333. https://doi.org/10.1111/j.1365-313X.2005.02530.x.

    • Search Google Scholar
    • Export Citation
  • Debnath SC. 2004. Clonal propagation of dwarf raspberry (Rubus pubescens Raf.) through in vitro axillary shoot proliferation. Plant Growth Regulat. 43:179186. https://doi.org/10.1023/B:GROW.0000040110.53216.6a.

    • Search Google Scholar
    • Export Citation
  • Debnath SC. 2005. A two-step procedure for adventitious shoot regeneration from in-vitro-derived lingonberry leaves: Shoot induction with TDZ and shoot elongation using Zea. HortScience. https://doi.org/10.21273/HORTSCI.40.1.189.

    • Search Google Scholar
    • Export Citation
  • Dewir YH, Nurmansyah Naidoo Y, Teixeira da Silva JA. 2018. Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Rep. 37:14511470. https://doi.org/10.1007/s00299-018-2326-1.

    • Search Google Scholar
    • Export Citation
  • Dirr MA, Heuser CW. 2006. The reference manual of woody plant propagation, from seed to tissue culture (2nd ed). Varsity Press, Inc., Athens, GA, USA.

  • Gardenia. 2019. Philadelphus microphyllus (little-leaf mockorange). Gardenia: Create Gardens. www.gardenia.net/plant/philadelphus-microphyllus. [accessed Sep 2023].

  • George EF, Hall MA, De Klerk G. 2007. Plant propagation by tissue culture: Volume 1. The Background. Springer Dordrecht, Exegetics, Basingstoke, UK. ISBN 978-1-4020-5005-3 (e-book). https://doi.org/10.1007/978-1-4020-5005-3.

  • Hare PD, Staden J, Van Staden J. 1994. Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus. Plant Cell Physiol. 35:11211125. https://doi.org/10.1093/oxfordjournals.pcp.a078704.

    • Search Google Scholar
    • Export Citation
  • Horgan R, Burch LR, Palni LMS. 1988. Cytokinin oxidase and the degradative metabolism of cytokinins, plant growth substances, p 282–290. In: Pharis RP, Rood SB (eds). Plant growth substances. Springer, Berlin, Heidelberg.

  • Jones MPA, Yi Z, Murch SJ, Saxena PK. 2007. Thidiazuron-induced regeneration of Echinacea purpurea L.: Micropropagation in solid and liquid culture systems. Plant Cell Rep. 26:1319. https://doi.org/10.1007/s00299-006-0209-3.

    • Search Google Scholar
    • Export Citation
  • Khajehyar R. 2021. Optimizing the culture medium for little-leaf mockorange (Philadelphus microphyllus) by using statistical modeling and spectral imaging (PhD Diss). University of Idaho, Moscow, ID, USA.

  • Khajehyar R, Tripepi RR. 2020. Different cytokinins and their concentrations affect shoot growth of little-leaf mockorange (Philadelphus microphyllus A. Gray) in tissue culture. Supplement to HortScience. ASHS 2020 Annual Conference. 55:366–367. (abstr.) https://doi.org/10.21273/HORTSCI.55.9S.S1.

  • Lady Bird Johnson Wildflower Center. 2015. Plant Database: Philadelphus microphyllus. Lady Bird Johnson Wildflower Center, The University of Texas at Austin. www.wildflower.org/plants/result.php?id_plant=phmi4. [accessed Sep 2023].

  • Lee ECM, Fossard RA. 1977. Some factors affecting multiple bud formation of strawberry (Fragaria ananassa Duchesne) in vitro. Acta Hortic. 78:187196. https://doi.org/10.17660/ActaHortic.1977.78.24.

    • Search Google Scholar
    • Export Citation
  • Loyola-Vargas VM, Vázquez-Flota F. 2006. An introduction to plant cell culture: Back to the future, p 3–8. In: Loyola-Vargas VM, Vázquez-Flota F (eds). Plant cell culture protocols. Methods in Molecular Biology™. vol. 318. Humana Press, Totowa, NJ, USA. https://doi.org/10.1385/1-59259-959-1:003.

  • Miller CO, Skoog F, Von Saltza MH, Strong FM. 1955. Kinetin, a cell division factor from deoxyribonucleic acid. J Am Chem Soc. 77:1392. https://doi.org/10.1021/ja01610a105.

    • Search Google Scholar
    • Export Citation
  • Miller RO, Gavlak R, Horneck D. 2013. Soil, plant and water reference methods for the Western region (4th ed). Western Region Extension Publication 125.

  • Mok MC, Mok DWS, Armstrong DJ. 1982. Cytokinin activity of N-phenyl-N-1, 2, 3-thidiiazol-5ylurea (TDZ). Phytochemistry. 21:15091511.

  • Mok MC, Mok DWS. 1985. The metabolism of [14C]-thidiazuron in callus tissues of Phaseolus lunatus. Physiol Plant. 65:427432. https://doi.org/10.1111/j.1399-3054.1985.tb08668.x.

    • Search Google Scholar
    • Export Citation
  • Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 15:473497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x.

    • Search Google Scholar
    • Export Citation
  • Murch SJ, Victor JMR, Krishnaraj S, Saxena PK. 1999. The role of proline in thidiazuron-induced somatic embryogenesis of peanut. In Vitro Cell Dev Biol Plant. 35:102105. https://doi.org/10.1007/s11627-999-0018-9.

    • Search Google Scholar
    • Export Citation
  • Najafian Ashrafi E, Vahdati K, Ebrahimzadeh H, Mirmasoumi M, Lotfi N. 2009. Optimization of walnut tissue culture medium using seed composition. Hortic Environ Biotechnol. 50:148153.

    • Search Google Scholar
    • Export Citation
  • Ostrolucká MG, Libiaková G, Ondrußková E, Gajdoßová A. 2004. In vitro propagation of Vaccinium species. Acta Universitatis Latviensis. Biology (Basel). 676:207212.

    • Search Google Scholar
    • Export Citation
  • Ramage CM, Williams RR. 2002. Mineral nutrition and plant morphogenesis. In Vitro Cell Dev Biol Plant. 38:116124. https://doi.org/10.1079/IVP2001269.

    • Search Google Scholar
    • Export Citation
  • Ramage CM. 1999. The role of mineral nutrients in the regulation of plant development in vitro (PhD Diss). The University of Queensland, Brisbane, Australia.

  • Rubio V, Bustos R, Irigoyen ML, Cardona-Lo’pez X, Rojas-Triana M, Paz-Are J. 2009. Plant hormones and nutrient signaling. Plant Mol Biol. 69:361373. https://doi.org/10.1007/s11103-008-9380-y.

    • Search Google Scholar
    • Export Citation
  • Sakakibara H, Takei K, Hirose N. 2006. Interactions between nitrogen and cytokinin in the regulation of metabolism and development. Trends Plant Sci. 11:440448. https://doi.org/10.1016/j.tplants.2006.07.004.

    • Search Google Scholar
    • Export Citation
  • SAS Institute Inc. 2016. SAS/ACCESS® 9.4 Interface to ADABAS: Reference. SAS Institute Inc., Cary, NC, USA.

  • Scheible WR, Morcuende R, Czechowski T, Fritz C, Osuna D, Palacios-Rojas N, Schindelasch D, Thimm OL, Udvardi MK, Stitt M. 2004. Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen. Plant Physiol. 136:24832499. https://doi.org/10.1104/pp.104.047019.

    • Search Google Scholar
    • Export Citation
  • Sen A, Batra A. 2011. Crucial role of nitrogen in in-vitro regeneration of Phyllanthus amarus Schum. and Thonn. Int J Pharm Sci Res. 2:21462151. https://doi.org/10.13040/IJPSR.0975-8232.2(8).2146-51.

    • Search Google Scholar
    • Export Citation
  • Skoog F, Miller CO. 1957. Chemical regulation of growth and organ formation in plant tissue cultured in vitro. Symp Soc Exp Biol. 11:118131.

    • Search Google Scholar
    • Export Citation
  • Smith RH. 2012. Plant tissue culture: Techniques and experiments. Elsevier Science, Academic Press, Cambridge, MA, USA. ISBN 9780124159853.

  • Stroup WW. 2014. Rethinking the analysis of non-normal data in plant and soil science. Agron J. 106:117. https://doi.org/10.2134/agronj2013.0342.

    • Search Google Scholar
    • Export Citation
  • Trigiano RN, Gray DJ. 2010. Plant tissue culture, development, and biotechnology (1st ed). CRC Press: Taylor & Francis Group, Boca Raton, FL, USA. ISBN 978-1-4200-8326-2.

  • Valero-Aracama C, Kane ME, Wilson SB, Philman NL. 2010. Substitution of benzyladenine with meta-topolin during shoot multiplication increases acclimatization of difficult- and easy-to-acclimatize sea oats (Uniola paniculate L.) genotypes. Plant Growth Regulat. 60:4349. https://doi.org/10.1007/s10725-009-9417-5.

    • Search Google Scholar
    • Export Citation
  • Wikipedia. 2019. Hydrangeaceae. https://en.wikipedia.org/wiki/Hydrangeaceae. [accessed Sep 2023].

  • Zhang CR, Huang XL, Wu JY, Feng BH, Chen YF. 2006. Identification of thidiazuron-induced ESTs expressed differentially during callus differentiation of alfalfa (Medicago sativa). Physiol Plant. 128:732739. https://doi.org/10.1111/j.1399-3054.2006.00763.x.

    • Search Google Scholar
    • Export Citation
  • Fig. 1.

    The selected little-leaf mockorange from Aberdeen Research and Extension Center, University of Idaho, Aberdeen, ID, USA (picture taken by Dr. Stephen Love).

  • Fig. 2.

    Effects of different cytokinins [benzylaminopurine (BA), kinetin (Kin), zeatin (Zea), meta-topolin (MT), dimethylamino purine (2iP), and thidiazuron (TDZ)] on in vitro growth of little-leaf mockorange shoots averaged over their concentrations for number of axillary shoots (A) and shoot length (B) of in vitro shoots. The interaction between cytokinins (BA, Zea, and TDZ) selected for their strong, positive, and negative effects and their concentrations on dry weight of little-leaf mockorange shoot cultures (C). Data are means (n = 4), and error bars indicate ± 95% confidence limits.

  • Fig. 3.

    The effects of different concentrations of MT on the number of axillary shoots formed by little-leaf mockorange shoot cultures (A). Comparison between different concentrations of MT (B) and BA (C) on shoot length of little-leaf mockorange cultures. The effects of different concentrations of TDZ on shoot length of little-leaf mockorange shoot cultures (D). The effect of 0, 0.55, 1.1, 1.65, or 2.2 µM zeatin on shoot length (E) and dry weight (F) of little-leaf mockorange. Data were means (n = 4), and error bars indicate ± 95% confidence limits.

  • Fig. 4.

    Effects of different N concentrations on number of axillary shoots. Data were means (n = 4), and error bars indicate ± 95% confidence limits. The ½ MS treatment (positive control) contained 30 mM N.

  • Fig. 5.

    Effects of different N concentrations (first experiment, A), Fe (first experiment, B; second experiment, C), Ca (D), Mg (E), and P (F) concentrations on shoot length of little-leaf mockorange in tissue culture. Data were means (n = 4), and error bars indicated ± 95% confidence limits. The ½ MS treatment (positive control) contained 30 mM N, 50 µM Fe, 1.5 mM Ca, 0.75 mM Mg, or 0.625 mM P in each individual mineral experiment.

  • Fig. 6.

    Effects of different N (first experiment, A; second experiment, B), Fe (first experiment, C; second experiment, D), Ca (E), and P (F) concentrations on dry weight of little-leaf mockorange in tissue culture. Data were means (n = 4), and error bars indicated ± 95% confidence limits. The ½ MS treatment (positive control) contained 30 mM N, 50 µM Fe, 1.5 mM Ca, 0.75 mM Mg, or 0.625 mM P in each individual mineral experiment.

  • Ali A, Ahmad T, Abbasi NA, Hafiz IA. 2009. Effect of different media and growth regulators on in vitro shoot proliferation of olive cultivar ‘Moraiolo’. Pak J Bot. 41:783795.

    • Search Google Scholar
    • Export Citation
  • Alosaimi AA, Tripepi RR. 2018. Micropropagation of Epilobium canum Garretti (firechalice) by axillary shoot culture. HortScience. 53:6266. https://doi.org/10.21273/HORTSCI12396-17.

    • Search Google Scholar
    • Export Citation
  • Arndt FR, Rusch R, Stillfried HV, Hanisch B, Martin WC. 1976. SN: 49537, a new defoliant (abstr). Plant Physiol. 57:99.

  • Bairu MW, Stirk WA, Dolezal K, Van Staden J. 2007. Optimizing the micropropagation protocol for the endangered Aloe polyphylla: Can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin? Plant Cell Tissue Organ Cult. 90:1523. https://doi.org/10.1007/s11240-007-9233-4.

    • Search Google Scholar
    • Export Citation
  • Brenner WG, Romanov GA, Köllmer I, Bürkle L, Schmülling T. 2005. Immediate-early and delayed cytokinin response genes of Arabidopsis thaliana identified by genome-wide expression profiling reveal novel cytokinin-sensitive processes and suggest cytokinin action through transcriptional cascades. Plant J. 44:314333. https://doi.org/10.1111/j.1365-313X.2005.02530.x.

    • Search Google Scholar
    • Export Citation
  • Debnath SC. 2004. Clonal propagation of dwarf raspberry (Rubus pubescens Raf.) through in vitro axillary shoot proliferation. Plant Growth Regulat. 43:179186. https://doi.org/10.1023/B:GROW.0000040110.53216.6a.

    • Search Google Scholar
    • Export Citation
  • Debnath SC. 2005. A two-step procedure for adventitious shoot regeneration from in-vitro-derived lingonberry leaves: Shoot induction with TDZ and shoot elongation using Zea. HortScience. https://doi.org/10.21273/HORTSCI.40.1.189.

    • Search Google Scholar
    • Export Citation
  • Dewir YH, Nurmansyah Naidoo Y, Teixeira da Silva JA. 2018. Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Rep. 37:14511470. https://doi.org/10.1007/s00299-018-2326-1.

    • Search Google Scholar
    • Export Citation
  • Dirr MA, Heuser CW. 2006. The reference manual of woody plant propagation, from seed to tissue culture (2nd ed). Varsity Press, Inc., Athens, GA, USA.

  • Gardenia. 2019. Philadelphus microphyllus (little-leaf mockorange). Gardenia: Create Gardens. www.gardenia.net/plant/philadelphus-microphyllus. [accessed Sep 2023].

  • George EF, Hall MA, De Klerk G. 2007. Plant propagation by tissue culture: Volume 1. The Background. Springer Dordrecht, Exegetics, Basingstoke, UK. ISBN 978-1-4020-5005-3 (e-book). https://doi.org/10.1007/978-1-4020-5005-3.

  • Hare PD, Staden J, Van Staden J. 1994. Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus. Plant Cell Physiol. 35:11211125. https://doi.org/10.1093/oxfordjournals.pcp.a078704.

    • Search Google Scholar
    • Export Citation
  • Horgan R, Burch LR, Palni LMS. 1988. Cytokinin oxidase and the degradative metabolism of cytokinins, plant growth substances, p 282–290. In: Pharis RP, Rood SB (eds). Plant growth substances. Springer, Berlin, Heidelberg.

  • Jones MPA, Yi Z, Murch SJ, Saxena PK. 2007. Thidiazuron-induced regeneration of Echinacea purpurea L.: Micropropagation in solid and liquid culture systems. Plant Cell Rep. 26:1319. https://doi.org/10.1007/s00299-006-0209-3.

    • Search Google Scholar
    • Export Citation
  • Khajehyar R. 2021. Optimizing the culture medium for little-leaf mockorange (Philadelphus microphyllus) by using statistical modeling and spectral imaging (PhD Diss). University of Idaho, Moscow, ID, USA.

  • Khajehyar R, Tripepi RR. 2020. Different cytokinins and their concentrations affect shoot growth of little-leaf mockorange (Philadelphus microphyllus A. Gray) in tissue culture. Supplement to HortScience. ASHS 2020 Annual Conference. 55:366–367. (abstr.) https://doi.org/10.21273/HORTSCI.55.9S.S1.

  • Lady Bird Johnson Wildflower Center. 2015. Plant Database: Philadelphus microphyllus. Lady Bird Johnson Wildflower Center, The University of Texas at Austin. www.wildflower.org/plants/result.php?id_plant=phmi4. [accessed Sep 2023].

  • Lee ECM, Fossard RA. 1977. Some factors affecting multiple bud formation of strawberry (Fragaria ananassa Duchesne) in vitro. Acta Hortic. 78:187196. https://doi.org/10.17660/ActaHortic.1977.78.24.

    • Search Google Scholar
    • Export Citation
  • Loyola-Vargas VM, Vázquez-Flota F. 2006. An introduction to plant cell culture: Back to the future, p 3–8. In: Loyola-Vargas VM, Vázquez-Flota F (eds). Plant cell culture protocols. Methods in Molecular Biology™. vol. 318. Humana Press, Totowa, NJ, USA. https://doi.org/10.1385/1-59259-959-1:003.

  • Miller CO, Skoog F, Von Saltza MH, Strong FM. 1955. Kinetin, a cell division factor from deoxyribonucleic acid. J Am Chem Soc. 77:1392. https://doi.org/10.1021/ja01610a105.

    • Search Google Scholar
    • Export Citation
  • Miller RO, Gavlak R, Horneck D. 2013. Soil, plant and water reference methods for the Western region (4th ed). Western Region Extension Publication 125.

  • Mok MC, Mok DWS, Armstrong DJ. 1982. Cytokinin activity of N-phenyl-N-1, 2, 3-thidiiazol-5ylurea (TDZ). Phytochemistry. 21:15091511.

  • Mok MC, Mok DWS. 1985. The metabolism of [14C]-thidiazuron in callus tissues of Phaseolus lunatus. Physiol Plant. 65:427432. https://doi.org/10.1111/j.1399-3054.1985.tb08668.x.

    • Search Google Scholar
    • Export Citation
  • Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 15:473497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x.

    • Search Google Scholar
    • Export Citation
  • Murch SJ, Victor JMR, Krishnaraj S, Saxena PK. 1999. The role of proline in thidiazuron-induced somatic embryogenesis of peanut. In Vitro Cell Dev Biol Plant. 35:102105. https://doi.org/10.1007/s11627-999-0018-9.

    • Search Google Scholar
    • Export Citation
  • Najafian Ashrafi E, Vahdati K, Ebrahimzadeh H, Mirmasoumi M, Lotfi N. 2009. Optimization of walnut tissue culture medium using seed composition. Hortic Environ Biotechnol. 50:148153.

    • Search Google Scholar
    • Export Citation
  • Ostrolucká MG, Libiaková G, Ondrußková E, Gajdoßová A. 2004. In vitro propagation of Vaccinium species. Acta Universitatis Latviensis. Biology (Basel). 676:207212.

    • Search Google Scholar
    • Export Citation
  • Ramage CM, Williams RR. 2002. Mineral nutrition and plant morphogenesis. In Vitro Cell Dev Biol Plant. 38:116124. https://doi.org/10.1079/IVP2001269.

    • Search Google Scholar
    • Export Citation
  • Ramage CM. 1999. The role of mineral nutrients in the regulation of plant development in vitro (PhD Diss). The University of Queensland, Brisbane, Australia.

  • Rubio V, Bustos R, Irigoyen ML, Cardona-Lo’pez X, Rojas-Triana M, Paz-Are J. 2009. Plant hormones and nutrient signaling. Plant Mol Biol. 69:361373. https://doi.org/10.1007/s11103-008-9380-y.

    • Search Google Scholar
    • Export Citation
  • Sakakibara H, Takei K, Hirose N. 2006. Interactions between nitrogen and cytokinin in the regulation of metabolism and development. Trends Plant Sci. 11:440448. https://doi.org/10.1016/j.tplants.2006.07.004.

    • Search Google Scholar
    • Export Citation
  • SAS Institute Inc. 2016. SAS/ACCESS® 9.4 Interface to ADABAS: Reference. SAS Institute Inc., Cary, NC, USA.

  • Scheible WR, Morcuende R, Czechowski T, Fritz C, Osuna D, Palacios-Rojas N, Schindelasch D, Thimm OL, Udvardi MK, Stitt M. 2004. Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen. Plant Physiol. 136:24832499. https://doi.org/10.1104/pp.104.047019.

    • Search Google Scholar
    • Export Citation
  • Sen A, Batra A. 2011. Crucial role of nitrogen in in-vitro regeneration of Phyllanthus amarus Schum. and Thonn. Int J Pharm Sci Res. 2:21462151. https://doi.org/10.13040/IJPSR.0975-8232.2(8).2146-51.

    • Search Google Scholar
    • Export Citation
  • Skoog F, Miller CO. 1957. Chemical regulation of growth and organ formation in plant tissue cultured in vitro. Symp Soc Exp Biol. 11:118131.

    • Search Google Scholar
    • Export Citation
  • Smith RH. 2012. Plant tissue culture: Techniques and experiments. Elsevier Science, Academic Press, Cambridge, MA, USA. ISBN 9780124159853.

  • Stroup WW. 2014. Rethinking the analysis of non-normal data in plant and soil science. Agron J. 106:117. https://doi.org/10.2134/agronj2013.0342.

    • Search Google Scholar
    • Export Citation
  • Trigiano RN, Gray DJ. 2010. Plant tissue culture, development, and biotechnology (1st ed). CRC Press: Taylor & Francis Group, Boca Raton, FL, USA. ISBN 978-1-4200-8326-2.

  • Valero-Aracama C, Kane ME, Wilson SB, Philman NL. 2010. Substitution of benzyladenine with meta-topolin during shoot multiplication increases acclimatization of difficult- and easy-to-acclimatize sea oats (Uniola paniculate L.) genotypes. Plant Growth Regulat. 60:4349. https://doi.org/10.1007/s10725-009-9417-5.

    • Search Google Scholar
    • Export Citation
  • Wikipedia. 2019. Hydrangeaceae. https://en.wikipedia.org/wiki/Hydrangeaceae. [accessed Sep 2023].

  • Zhang CR, Huang XL, Wu JY, Feng BH, Chen YF. 2006. Identification of thidiazuron-induced ESTs expressed differentially during callus differentiation of alfalfa (Medicago sativa). Physiol Plant. 128:732739. https://doi.org/10.1111/j.1399-3054.2006.00763.x.

    • Search Google Scholar
    • Export Citation
Razieh Khajehyar Department of Plant Sciences, University of Idaho, Moscow, ID 83844, USA

Search for other papers by Razieh Khajehyar in
Google Scholar
Close
,
Robert Tripepi Department of Plant Sciences, University of Idaho, Moscow, ID 83844, USA

Search for other papers by Robert Tripepi in
Google Scholar
Close
,
Stephen Love Department of Plant Sciences, University of Idaho, Moscow, ID 83844, USA

Search for other papers by Stephen Love in
Google Scholar
Close
, and
William J. Price Department of Mathematics and Statistical Sciences, University of Idaho, Moscow, ID 83844, USA

Search for other papers by William J. Price in
Google Scholar
Close

Contributor Notes

R.Z. is the corresponding author. E-mail: rkhajehyar@yahoo.com.

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 915 915 34
PDF Downloads 179 179 27
Save