Plant in vitro organogenesis is well-controlled and thus provides an ideal system for plant propagation and studying mechanisms of plant development. However, the data on systematic in vitro organogenesis from leaf explant with various concentrations and combinations of hormones are limited. Arabidopsis is a very useful model plant species for many aspects of plant biological study. Here, we reported a simple, fast and efficient one-step process for evaluating leaf explant-derived in vitro Arabidopsis organogenesis involving the application of various concentrations and combinations of exogenous hormones. The central portion of the fourth rosette leaf was harvested from the 21-days-old seedling and cultured in vitro on the media containing 216 combinations of exogenous hormones. Different types of organs, including roots, shoots, inflorescences, and leaf-like organs were initiated from leaf explants. Several optimal experimental combinations were selected. A hormone combination, 1.00 μM NAA + 10.00 μM ZT, was considered as the most efficient one for adventitious shoot regeneration. And for adventitious root regeneration, six hormone combinations, [(NAA + ZT: 1.00 + 0.10 μM; 10.00 + 0.01 μM; 20.00 + 0.10 μM; 20.00 + 1.00 μM) and (NAA + 6-BA: 10.00 + 0.10 μM; 20.00 + 10.00 μM)], were thought to be the best ones. Further, both auxin and cytokinin ratios and concentrations were crucial for efficient in vitro organogenesis. Our study provides the important information for hormone-regulated organogenesis.
Plant cells, organs and tissues exhibit a remarkable ability to regenerate new organs under in vitro conditions [
Exogenous hormones are widely regarded as being more important than other factors [
It has been shown that in some plants semi-mature and fully developed leaves gave good response of shoot organogenesis in vitro [
Arabidopsis is a very useful model plant species for developmental studies, biological investigations and genetic engineering due to its unique genetic and developmental characteristics. Recent advances in genetic and molecular techniques, combined with unique approaches to tissue culture, may provide new insights into the mechanism underlying many fundamental biological activities such as cell dedifferentiation, cell developmental fate determination and intercellular communication [
In this study, we describe a simple, fast and efficient one-step process for organogenesis from leaf-derived callus and present the effects of various concentrations of exogenous hormones on in vitro organogenesis in Arabidopsis. In our previous work, the one-step process for in vitro organ regeneration had been developed and defined [
Arabidopsis Wassilewskija-0 (WS-0) (Provided by the Arabidopsis Biological Resource Center) ecotype seeds were surface sterilized with 70% (v/v) ethanol for 5 min followed by 2.6% (v/v) commercial bleach with a drop of tween-20 (Sigma-Aldrich) for 10 min, and then washed four times with sterile ddH2O prior to plating on half-strength Murashige and Skoog (MS) medium (Sigma-aldarich, pH5.8, 1L , consisting of 0.825 g NH4NO3, 0.95 g KNO3, 0.22 g CaCl2∙2H2O, 0.185 g MgSO4∙7H2O, 0.085 g KH2PO4, 0.83 mg KI, 6.2 mg H3BO3, 22.3 mg MnSO4∙4H2O, 8.6 mg ZnSO4∙7H2O, 0.25 mg Na2MoO4∙2H2O, 0.025 mg CuSO4∙5H2O, 0.025 mg CoCl2∙6H2O, 37.3 mg FeSO4∙7H2O, 27.8 mg Na2-EDTA∙2H2O, 100 mg inositol, 0.1 mg VB1, 0.5 mg VB3, 0.5 mg VB6, 2.0 mg glycine. Murashige and Skoog, 1962) [
The primary goal of this study was to characterize the effects of exogenous hormones on organogenesis initiated from leaf explants. Systematic changes in auxin and cytokinin concentrations were designed and used to establish a one-step approach for callus induction as well as organ regeneration. This one step approach includes the use of the MS basal medium (Sigma-aldarich, pH5.8, 1L , consisting of 1.65 g NH4NO3, 1.9 g KNO3, 0.44 g CaCl2∙2H2O, 0.37 g MgSO4∙7H2O, 0.17 g KH2PO4, 0.83 mg KI, 6.2 mg H3BO3, 22.3 mg MnSO4∙4H2O, 8.6 mg ZnSO4∙7H2O, 0.25 mg Na2MoO4∙2H2O, 0.025 mg CuSO4∙5H2O, 0.025 mg CoCl2∙6H2O, 37.3 mg FeSO4∙7H2O, 27.8 mg Na2-EDTA∙2H2O, 100 mg inositol, 0.1 mg VB1, 0.5 mg VB3, 0.5 mg VB6, 2.0 mg glycine) [
The pH value of media mentioned above was adjusted to 5.8 using pH meter (UB-7, Denver Instrument Company, USA) before autoclaving for 20 minutes at 121˚C, 105 kPa.
All kinds of hormones were produced by Sigma-Aldrich. One hundred milligram IAA (No. 12886), 2,4-D (D7299) or NAA (N0640) was dissolved in a small volume of ethyl alcohol, and then brought to 1 ml with ddH2O. For cytokinin, a few drops of 1 NaOH were required to bring ZT (100 mg) or 6-BA (100 mg) into solution, and then ddH2O was added up to 100 ml. Filtration was required for ZT solution because it was considered to be unstable in the heat. The stock solutions of NAA and 2,4-D were stored at 4˚C, and IAA, ZT, and 6-BA were stored at −20˚C.
Morphological observations of regenerated organs were conducted using an Olympus SZX16 stereo microscope (Olympus America Inc.). Microscopy images were captured using an Olympus DP72 digital camera (Olympus America Inc.).
The frequency of callus induction and number of calli induced from explants was assessed at approximately experimental day 7. Evaluation of explant regeneration efficiency began after one to two weeks in culture. The parameters investigated in the present study include the regeneration frequency (% of explants capable of organ regeneration under the present in vitro conditions) of adventitious shoots, inflorescences, leaf-like organs, and adventitious roots. Regeneration frequency was calculated as the percentage of explants capable of organ regeneration under the culture conditions described above. Equation is as following:
Data collected from each of three replicate experiments were averaged and expressed as the mean ± s.e.m.
Leaf explant-derived plant regeneration has been widely used for various studies conducted in many plant species [
Efficient callus induction was observed on all sides of the wounded explant, especially on the midrib, within two weeks in culture (
Among the four types of regenerated organs from leaf explants, adventitious root regeneration was the most frequent. Leaf explants responded well to nearly 50% of the designed media containing various hormone combinations [(NAA + ZT: 1.00 + 0.10 μM; 10.00 + 0.01 μM; 20.00 + 0.10 μM; 20.00 + 1.00 μM) or (NAA + 6-BA: 10.00 + 0.10 μM; 20.00 + 10.00 μM)], generating the highest frequency (100%) of adventitious root formation
(
In the present study, different organ types were induced from explants with varying frequency, depending on the culture conditions (
To address this topic, six concentrations per hormone were devised in order to determine the best concentration range based on the response of explant cells. As shown in
Optimal concentrations of IAA for effective shoot induction ranged from 0.01 to 1.00 μM, followed by optimal concentrations of NAA (0.01 to 20.00 μM) and 2,4-Dichlorophenoxyacetic acid (2,4-D) (0.01-1.00 μM). High shoot regeneration frequency occurred when these were combined with 1.00 to 20.00 μM of 6-BA or with 0.10 to 20.00 μM of ZT (
Regenerated organs | Frequency (%) | |
---|---|---|
Shoots | Meana | 5.49 |
Maximumb | 80.00 ± 4.58 | |
Inflorescences | Meanc | 0.49 |
Maximumd | 55.30 ± 2.65 | |
Leaf-like organs | Meane | 1.56 |
Maximumf | 21.22 ± 4.02 | |
Roots | Meang | 21.89 |
Maximumh | 97.07 ± 2.11 |
aThe number represents the mean arithmetical value of the frequencies of regenerated shoots. bThe number represents the highest frequency of regenerated shoots. cThe number represents the mean arithmetical value of the frequencies of regenerated inflorescences. dThe number represents the highest frequency of regenerated inflorescences. eThe number represents the mean arithmetical value of the frequencies of regenerated leaf-like organs. fThe number represents the highest frequency of regenerated leaf-like organs. gThe number represents the mean arithmetical value of the frequencies of regenerated roots. h The number represents the highest frequency of regenerated roots.
Type of Regenerated organs | Combination (Auxin/CK) | Concentration (μM) | Regenerated Frequency (%) |
---|---|---|---|
Shoots | 2,4-D/6-BA | 0.10/10.00 | 81.25 ± 7.02 |
2,4-D/ZT | 0.10/10.00 | 91.30 ± 5.70 | |
IAA/6-BA | 0.10/10.00 | 19.71 ± 1.65 | |
IAA/ZT | 10.00/20.00 | 62.56 ± 3.42 | |
NAA/6-BA | 1.00/10.00 | 35.78 ± 2.54 | |
NAA/ZT | 1.00/10.00 | 80.00 ± 4.58 | |
Roots | |||
2,4-D/6-BA | 0.10/0.10 | 88.25 ± 5.34 | |
2,4-D/ZT | 0.01/0.00 | 54.50 ± 2.51 | |
IAA/6-BA | 20.00/0.00 | 93.34 ± 2.74 | |
IAA/ZT | 10.00/0.01 | 96.65 ± 0.86 | |
NAA/6-BA | 10.00/0.10 | 100.00 | |
NAA/6-BA | 20.00/10.00 | 100.00 | |
NAA/ZT | 1.00/0.10 | 100.00 | |
NAA/ZT | 10.00/0.01 | 100.00 | |
NAA/ZT | 20.00/0.10 | 100.00 | |
NAA/ZT | 20.00/1.00 | 100.00 |
both the hormone ratios and their concentrations are crucial for deter mining cell developmental fate during in vitro organogenesis.
In the present study, IAA (natural auxin) was the most efficient auxin, whereas 2,4-D (synthetic auxin) was the least effective auxin at inducing leaf explant-derived shoot regeneration (Figures 2(A)-(F)). A wide range of NAA (synthetic auxin) concentrations was capable of inducing adventitious roots at very high efficiencies compared to IAA and 2,4-D (
Another important observation revealed by the present study was that auxin alone could initiate callus and/or adventitious root formation and sometimes shoot formation from each of the three types of explants with variable induction rates (
Additionally, this study has also demonstrated that the use of cytokinin (e.g., ZT or 6-BA) at a lower concentration efficiently induced adventitious root formation (Figures 2(G)-(L)). A similar finding has been reported by George et al. (2008) [
cytokinin concentrations (Figures 2(G)-(L)). The ZT-treated leaf explants that had undergone a reduced induction period generated a higher rate of organogenesis than did the 6-BA-treated leaf explants (Figures 2(A)-(F)). These results indicate that the natural cytokinin ZT might be more efficient than the synthetic cytokinin 6-BA in triggering Arabidopsis leaf explant-derived shoot initiation.
This research is supported by grants from the National Natural Science Foundation of China (90917015, 31000652) and the Ministry of Science and Technology (MOST) of China (2007CB948200).
Cytokinin 6-BA or ZT (μM) | |||||||
---|---|---|---|---|---|---|---|
0.00 | 0.01 | 0.10 | 1.00 | 10.00 | 20.00 | ||
Auxin IAA or NAA or 2,4-D (μM) | 0.00 | (0.00, 0.00)* | (0.00, 0.01) | (0.00, 0.10) | (0.00, 1.00) | (0.00, 10.00) | (0.00, 20.00) |
0.01 | (0.01, 0.00) | (0.01, 0.01) | (0.01, 0.10) | (0.00, 1.00) | (0.01, 10.00) | (0.01, 20.00) | |
0.10 | (0.10, 0.00) | (0.10, 0.01) | (0.10, 0.10) | (0.00, 1.00) | (0.10, 10.00) | (0.10, 20.00) | |
1.00 | (1.00, 0.00) | (1.00, 0.01) | (1.00, 0.10) | (0.00, 1.00) | (1.00, 10.00) | (1.00, 20.00) | |
10.00 | (10.00, 0.00) | (10.00, 0.01) | (10.00, 0.10) | (10.00, 1.00) | (10.00, 10.00) | (10.00, 20.00) | |
20.00 | (20.00, 0.00) | (20.00, 0.01) | (20.00, 0.10) | (20.00, 1.00) | (20.00, 10.00) | (20.00, 20.00) |
Exogenous Hormones | Organ Regeneration Frequency (%) | |||||
---|---|---|---|---|---|---|
Combination (Auxin/CK) | Concentration (μM) | Calli | Adventitious Shoots | Inflorescences | Leaf-Like Organs | Adventitious Roots |
2,4-D/6-BA | 0.00/0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 0.00/0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 0.00/0.10 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 0.00/1.00 | 21.55 ± 2.46 | 6.76 ± 1.52 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 0.00/10.00 | 31.57 ± 1.54 | 5.38 ± 1.69 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 0.00/20.00 | 31.64 ± 1.98 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 0.01/0.00 | 75.70 ± 2.59 | 0.00 | 0.00 | 0.00 | 54.55 ± 4.52 |
2,4-D/6-BA | 0.01/0.01 | 70.62 ± 1.24 | 0.00 | 0.00 | 0.00 | 35.32 ± 1.35 |
2,4-D/6-BA | 0.01/0.10 | 87.90 ± 3.26 | 0.00 | 0.00 | 0.00 | 66.77 ± 3.54 |
2,4-D/6-BA | 0.01/1.00 | 46.34 ± 2.57 | 0.00 | 0.00 | 0.00 | 31.39 ± 1.30 |
2,4-D/6-BA | 0.01/10.00 | 33.30 ± 3.36 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 0.01/20.00 | 2.95 ± 1.20 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 0.10/0.00 | 37.64 ± 5.34 | 0.00 | 0.00 | 0.00 | 31.38 ± 2.30 |
2,4-D/6-BA | 0.10/0.01 | 76.56 ± 1.49 | 0.00 | 0.00 | 0.00 | 35.35 ± 3.52 |
2,4-D/6-BA | 0.10/0.10 | 91.16 ± 3.65 | 10.30 ± 1.35 | 2.92 ± 0.32 | 0.00 | 88.25 ± 5.34 |
2,4-D/6-BA | 0.10/1.00 | 100.00 | 79.35 ± 4.32 | 0.00 | 7.85 ± 0.75 | 20.07 ± 2.10 |
2,4-D/6-BA | 0.10/10.00 | 56.38 ± 3.54 | 81.25 ± 7.02 | 0.00 | 5.20 ± 1.31 | 0.00 |
2,4-D/6-BA | 0.10/20.00 | 18.44 ± 2.54 | 0.00 | 55.30 ± 2.65 | 0.00 | 0.00 |
2,4-D/6-BA | 1.00/0.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 1.00/0.01 | 97.25 ± 1.48 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 1.00/0.10 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 1.00/1.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
---|---|---|---|---|---|---|
2,4-D/6-BA | 1.00/10.00 | 93.88 ± 3.56 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 1.00/20.00 | 97.28 ± 2.54 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 10.00/0.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 10.00/0.01 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 10.00/0.10 | 97.29 ± 2.43 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 10.00/1.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 10.00/10.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 10.00/20.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 20.00/0.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 20.00/0.01 | 86.18 ± 1.64 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 20.00/0.10 | 48.58 ± 2.54 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 20.00/1.00 | 96.85 ± 1.59 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 20.00/10.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/6-BA | 20.00/20.00 | 91.79 ± 1.63 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 0.00/0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 0.00/0.01 | 50.00 ± 2.15 | 0.00 | 0.00 | 0.00 | 3.33 ± 2.35 |
2,4-D/ZT | 0.00/0.10 | 50.80 ± 2.45 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 0.00/1.00 | 58.65 ± 1.58 | 6.90 ± 2.96 | 0.00 | 4.52 ± 1.03 | 3.45 ± 2.03 |
2,4-D/ZT | 0.00/10.00 | 85.71 ± 4.23 | 7.14 ± 2.41 | 0.00 | 4.69 ± 1.54 | 0.00 |
2,4-D/ZT | 0.00/20.00 | 88.25 ± 1.62 | 14.29 ± 1.65 | 0.00 | 5.21 ± 2.01 | 0.00 |
2,4-D/ZT | 0.01/0.00 | 75.58 ± 0.87 | 0.00 | 0.00 | 0.00 | 54.50 ± 2.51 |
2,4-D/ZT | 0.01/0.01 | 25.52 ± 2.31 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 0.01/0.10 | 23.53 ± 3.54 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 0.01/1.00 | 43.75 ± 2.46 | 3.00 ± 0.36 | 0.00 | 0.00 | 6.25 ± 1.03 |
2,4-D/ZT | 0.01/10.00 | 53.85 ± 2.78 | 30.77 ± 1.65 | 0.00 | 8.26 ± 1.05 | 0.00 |
2,4-D/ZT | 0.01/20.00 | 86.11 ± 4.18 | 22.22 ± 4.35 | 0.00 | 7.56 ± 1.54 | 0.00 |
2,4-D/ZT | 0.10/0.00 | 90.91 ± 5.14 | 0.00 | 0.00 | 0.00 | 42.42 ± 4.01 |
2,4-D/ZT | 0.10/0.01 | 40.00 ± 4.35 | 6.98 ± 1.25 | 0.00 | 0.00 | 8.57 ± 1.58 |
2,4-D/ZT | 0.10/0.10 | 100.00 | 15.70 ± 0.38 | 0.00 | 0.00 | 43.33 ± 3.26 |
2,4-D/ZT | 0.10/1.00 | 96.67 ± 2.65 | 90.60 ± 4.36 | 0.00 | 11.02 ± 1.33 | 6.67 ± 2.52 |
2,4-D/ZT | 0.10/10.00 | 93.75 ± 1.54 | 91.30 ± 5.70 | 0.00 | 9.78 ± 2.45 | 0.00 |
2,4-D/ZT | 0.10/20.00 | 100.00 | 82.60 ± 5.06 | 0.00 | 3.58 ± 0.65 | 10.81 ± 4.36 |
2,4-D/ZT | 1.00/0.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 1.00/0.01 | 91.45 ± 4.25 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 1.00/0.10 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 1.00/1.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 1.00/10.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
---|---|---|---|---|---|---|
2,4-D/ZT | 1.00/20.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 10.00/0.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 10.00/0.01 | 94.15 ± 2.85 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 10.00/0.10 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 10.00/1.00 | 96.96 ± 3.26 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 10.00/10.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 10.00/20.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 20.00/0.00 | 95.88 ± 1.25 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 20.00/0.01 | 87.55 ± 4.32 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 20.00/0.10 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 20.00/1.00 | 91.47 ± 1.58 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 20.00/10.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
2,4-D/ZT | 20.00/20.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.00/0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.00/0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.00/0.10 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.00/1.00 | 20.00 ± 2.97 | 6.77 ± 1.29 | 0.00 | 6.75 ± 4.31 | 0.00 |
IAA/6-BA | 0.00/10.00 | 31.65 ± 3.65 | 5.35 ± 1.54 | 0.00 | 5.34 ± 2.56 | 0.00 |
IAA/6-BA | 0.00/20.00 | 30.00 ± 2.65 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.01/0.00 | 5.62 ± 1.35 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.01/0.01 | 3.24 ± 2.11 | 0.00 | 0.00 | 0.00 | 3.23 ± 2.67 |
IAA/6-BA | 0.01/0.10 | 3.32 ± 2.30 | 0.00 | 0.00 | 0.00 | 3.35 ± 2.58 |
IAA/6-BA | 0.01/1.00 | 8.63 ± 2.01 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.01/10.00 | 21.91 ± 2.35 | 13.15 ± 0.69 | 0.00 | 1.50 ± 0.54 | 0.00 |
IAA/6-BA | 0.01/20.00 | 50.00 ± 2.41 | 3.12 ± 0.32 | 0.00 | 2.16 ± 0.63 | 0.00 |
IAA/6-BA | 0.10/0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.10/0.01 | 3.40 ± 0.67 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.10/0.10 | 5.00 ± 0.54 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 0.10/1.00 | 13.34 ± 2.34 | 0.00 | 0.00 | 0.00 | 13.35 ± 1.63 |
IAA/6-BA | 0.10/10.00 | 33.40 ± 1.25 | 19.71 ± 1.65 | 0.00 | 4.21 ± 1.03 | 0.00 |
IAA/6-BA | 0.10/20.00 | 60.00 ± 2.65 | 3.32 ± 0.25 | 0.00 | 2.25 ± 0.58 | 0.00 |
IAA/6-BA | 1.00/0.00 | 53.31 ± 2.54 | 0.00 | 0.00 | 0.00 | 53.30 ± 4.20 |
IAA/6-BA | 1.00/0.01 | 25.84 ± 0.64 | 0.00 | 0.00 | 0.00 | 16.10 ± 0.96 |
IAA/6-BA | 1.00/0.10 | 3.25 ± 0.58 | 0.00 | 0.00 | 0.00 | 3.20 ± 2.14 |
IAA/6-BA | 1.00/1.00 | 6.89 ± 1.32 | 3.80 ± 0.63 | 0.00 | 3.10 ± 1.12 | 3.08 ± 1.65 |
IAA/6-BA | 1.00/10.00 | 60.67 ± 2.58 | 6.10 ± 2.12 | 0.00 | 6.16 ± 1.45 | 0.00 |
---|---|---|---|---|---|---|
IAA/6-BA | 1.00/20.00 | 64.38 ± 1.45 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 10.00/0.00 | 47.19 ± 1.58 | 0.00 | 0.00 | 0.00 | 47.18 ± 5.98 |
IAA/6-BA | 10.00/0.01 | 62.95 ± 2.34 | 0.00 | 0.00 | 0.00 | 62.97 ± 3.45 |
IAA/6-BA | 10.00/0.10 | 86.79 ± 1.25 | 0.00 | 0.00 | 0.00 | 86.78 ± 1.54 |
IAA/6-BA | 10.00/1.00 | 66.80 ± 2.58 | 15.36 ± 3.15 | 0.00 | 12.5 ± 2.75 | 3.01 ± 0.68 |
IAA/6-BA | 10.00/10.00 | 64.90 ± 3.62 | 13.57 ± 2.10 | 0.00 | 10.35 ± 2.05 | 0.00 |
IAA/6-BA | 10.00/20.00 | 70.34 ± 1.54 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/6-BA | 20.00/0.00 | 93.31 ± 1.35 | 0.00 | 0.00 | 0.00 | 93.34 ± 2.74 |
IAA/6-BA | 20.00/0.01 | 87.15 ± 0.35 | 0.00 | 0.00 | 0.00 | 87.15 ± 2.51 |
IAA/6-BA | 20.00/0.10 | 90.38 ± 0.85 | 0.00 | 0.00 | 0.00 | 90.38 ± 3.46 |
IAA/6-BA | 20.00/1.00 | 99.95 ± 0.04 | 0.00 | 0.00 | 15.74 ± 0.86 | 17.64 ± 1.42 |
IAA/6-BA | 20.00/10.00 | 93.58 ± 2.36 | 0.00 | 0.00 | 7.62 ± 0.54 | 3.24 ± 0.56 |
IAA/6-BA | 20.00/20.00 | 93.56 ± 2.54 | 0.00 | 0.00 | 2.56 ± 1.35 | 0.00 |
IAA/ZT | 0.00/0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/ZT | 0.00/0.01 | 50.00 ± 1.48 | 0.00 | 0.00 | 0.00 | 3.33 ± 2.65 |
IAA/ZT | 0.00/0.10 | 58.62 ± 2.35 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/ZT | 0.00/1.00 | 75.00 ± 1.24 | 0.00 | 0.00 | 0.00 | 3.45 ± 3.21 |
IAA/ZT | 0.00/10.00 | 100.00 | 7.17 ± 1.36 | 0.00 | 0.00 | 0.00 |
IAA/ZT | 0.00/20.00 | 91.30 ± 3.51 | 0.00 | 0.00 | 0.00 | 3.16 ± 2.58 |
IAA/ZT | 0.01/0.00 | 5.60 ± 2.45 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/ZT | 0.01/0.01 | 27.90 ± 1.45 | 0.00 | 0.00 | 0.00 | 6.56 ± 2.51 |
IAA/ZT | 0.01/0.10 | 50.0 ± 2.36 | 0.00 | 0.00 | 0.00 | 3.13 ± 3.01 |
IAA/ZT | 0.01/1.00 | 80.00 ± 4.32 | 9.75 ± 2.41 | 0.00 | 0.00 | 0.00 |
IAA/ZT | 0.01/10.00 | 96.55 ± 1.35 | 12.96 ± 2.83 | 0.00 | 0.00 | 0.00 |
IAA/ZT | 0.01/20.00 | 88.89 ± 5.60 | 21.93 ± 1.75 | 0.00 | 5.82 ± 1.36 | 0.00 |
IAA/ZT | 0.10/0.00 | 32.14 ± 2.54 | 0.00 | 0.00 | 0.00 | 53.35 ± 2.98 |
IAA/ZT | 0.10/0.01 | 66.67 ± 1.55 | 0.00 | 0.00 | 0.00 | 17.25 ± 2.45 |
IAA/ZT | 0.10/0.10 | 72.41 ± 3.58 | 0.00 | 0.00 | 0.00 | 6.38 ± 1.56 |
IAA/ZT | 0.10/1.00 | 80.85 ± 4.65 | 0.00 | 0.00 | 0.00 | 0.00 |
IAA/ZT | 0.10/10.00 | 68.75 ± 1.75 | 12.19 ± 2.67 | 0.00 | 5.62 ± 2.31 | 0.00 |
IAA/ZT | 0.10/20.00 | 66.67 ± 2.85 | 23.38 ± 3.46 | 0.00 | 4.85 ± 1.52 | 0.00 |
IAA/ZT | 1.00/0.00 | 47.19 ± 2.45 | 0.00 | 0.00 | 0.00 | 47.18 ± 1.50 |
IAA/ZT | 1.00/0.01 | 80.05 ± 3.69 | 0.00 | 0.00 | 0.00 | 80.05 ± 3.48 |
IAA/ZT | 1.00/0.10 | 99.47 ± 1.85 | 0.00 | 0.00 | 0.00 | 93.15 ± 2.76 |
IAA/ZT | 1.00/1.00 | 33.39 ± 1.45 | 0.00 | 0.00 | 0.00 | 46.45 ± 2.55 |
IAA/ZT | 1.00/10.00 | 40.58 ± 2.35 | 11.92 ± 2.04 | 0.00 | 7.20 ± 2.98 | 0.00 |
---|---|---|---|---|---|---|
IAA/ZT | 1.00/20.00 | 100.00 | 34.30 ± 4.31 | 0.00 | 5.24 ± 2.50 | 0.00 |
IAA/ZT | 10.00/0.00 | 93.3 ± 1.24 | 0.00 | 0.00 | 0.00 | 93.36 ± 1.20 |
IAA/ZT | 10.00/0.01 | 99.91 ± 0.05 | 0.00 | 0.00 | 0.00 | 96.65 ± 0.86 |
IAA/ZT | 10.00/0.10 | 92.95 ± 2.61 | 6.98 ± 0.65 | 0.00 | 0.00 | 86.00 ± 1.02 |
IAA/ZT | 10.00/1.00 | 88.20 ± 2.53 | 10.78 ± 1.35 | 3.68 ± 0.87 | 0.00 | 31.05 ± 1.32 |
IAA/ZT | 10.00/10.00 | 85.74 ± 2.37 | 50.05 ± 2.85 | 0.00 | 12.57 ± 1.53 | 0.00 |
IAA/ZT | 10.00/20.00 | 95.85 ± 1.42 | 62.56 ± 3.42 | 0.00 | 21.22 ± 4.02 | 0.00 |
IAA/ZT | 20.00/0.00 | 83.87 ± 1.85 | 0.00 | 0.00 | 0.00 | 83.87 ± 4.25 |
IAA/ZT | 20.00/0.01 | 89.29 ± 1.54 | 0.00 | 0.00 | 0.00 | 71.43 ± 3.62 |
IAA/ZT | 20.00/0.10 | 83.87 ± 1.58 | 0.00 | 0.00 | 0.00 | 64.52 ± 5.32 |
IAA/ZT | 20.00/1.00 | 87.10 ± 1.62 | 8.35 ± 2.15 | 0.00 | 15.76 ± 1.17 | 22.58 ± 4.21 |
IAA/ZT | 20.00/10.00 | 93.10 ± 1.40 | 7.28 ± 1.34 | 3.47 ± 5.30 | 4.01 ± 2.08 | 0.00 |
IAA/ZT | 20.00/20.00 | 92.59 ± 2.36 | 21.47 ± 2.34 | 0.00 | 7.18 ± 1.62 | 3.15 ± 2.39 |
NAA/6-BA | 0.00/0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.00/0.01 | 25.75 ± 2.15 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.00/0.10 | 18.75 ± 1.45 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.00/1.00 | 85.71 ± 2.31 | 6.75 ± 2.31 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.00/10.00 | 75.86 ± 1.42 | 5.37 ± 1.36 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.00/20.00 | 75.96 ± 1.35 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.01/0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.01/0.01 | 17.86 ± 2.04 | 0.00 | 0.00 | 0.00 | 7.17 ± 1.58 |
NAA/6-BA | 0.01/0.10 | 47.06 ± 1.34 | 5.95 ± 2.14 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.01/1.00 | 42.86 ± 2.15 | 0.00 | 0.00 | 0.00 | 14.34 ± 1.14 |
NAA/6-BA | 0.01/10.00 | 51.52 ± 1.24 | 9.10 ± 2.04 | 0.00 | 2.31 ± 1.63 | 0.00 |
NAA/6-BA | 0.01/20.00 | 51.61 ± 2.45 | 3.20 ± 1.32 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.10/0.00 | 26.83 ± 1.35 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.10/0.01 | 92.65 ± 0.95 | 0.00 | 0.00 | 0.00 | 37.00 ± 4.35 |
NAA/6-BA | 0.10/0.10 | 77.59 ± 1.57 | 0.00 | 0.00 | 0.00 | 19.40 ± 2.15 |
NAA/6-BA | 0.10/1.00 | 56.41 ± 2.34 | 0.00 | 0.00 | 0.00 | 12.00 ± 2.63 |
NAA/6-BA | 0.10/10.00 | 93.87 ± 1.63 | 12.50 ± 1.25 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 0.10/20.00 | 61.36 ± 0.96 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/6-BA | 1.00/0.00 | 70.73 ± 1.54 | 0.00 | 0.00 | 0.00 | 46.75 ± 1.45 |
NAA/6-BA | 1.00/0.01 | 81.37 ± 1.42 | 0.00 | 0.00 | 0.00 | 50.08 ± 1.24 |
NAA/6-BA | 1.00/0.10 | 86.76 ± 1.36 | 0.00 | 0.00 | 0.00 | 86.70 ± 2.31 |
NAA/6-BA | 1.00/1.00 | 67.84 ± 2.54 | 10.75 ± 1.36 | 0.00 | 0.00 | 32.10 ± 2.12 |
NAA/6-BA | 1.00/10.00 | 89.39 ± 1.43 | 35.78 ± 2.54 | 0.00 | 2.58 ± 0.63 | 3.65 ± 1.20 |
---|---|---|---|---|---|---|
NAA/6-BA | 1.00/20.00 | 94.45 ± 1.35 | 20.67 ± 1.43 | 0.00 | 2.35 ± 0.85 | 20.65 ± 2.46 |
NAA/6-BA | 10.00/0.00 | 92.97 ± 1.59 | 0.00 | 0.00 | 0.00 | 92.98 ± 2.01 |
NAA/6-BA | 10.00/0.01 | 90.53 ± 1.54 | 0.00 | 0.00 | 0.00 | 90.05 ± 1.84 |
NAA/6-BA | 10.00/0.10 | 100.00 | 0.00 | 0.00 | 0.00 | 100.00 |
NAA/6-BA | 10.00/1.00 | 80.00 ± 2.35 | 0.00 | 0.00 | 0.00 | 80.98 ± 5.62 |
NAA/6-BA | 10.00/10.00 | 57.24 ± 1.24 | 14.35 ± 1.42 | 0.00 | 3.45 ± 0.78 | 42.95 ± 4.85 |
NAA/6-BA | 10.00/20.00 | 92.99 ± 1.85 | 17.97 ± 1.79 | 0.00 | 7.28 ± 0.80 | 39.34 ± 3.67 |
NAA/6-BA | 20.00/0.00 | 94.10 ± 1.24 | 0.00 | 5.85 ± 2.01 | 0.00 | 94.17 ± 2.35 |
NAA/6-BA | 20.00/0.01 | 92.24 ± 1.35 | 0.00 | 0.00 | 0.00 | 92.29 ± 3.24 |
NAA/6-BA | 20.00/0.10 | 97.04 ± 2.04 | 0.00 | 0.00 | 0.00 | 97.07 ± 2.11 |
NAA/6-BA | 20.00/1.00 | 94.25 ± 1.25 | 6.72 ± 3.21 | 0.00 | 0.00 | 87.58 ± 3.45 |
NAA/6-BA | 20.00/10.00 | 100.00 | 5.39 ± 1.35 | 25.8 ± 2.31 | 0.00 | 100.00 |
NAA/6-BA | 20.00/20.00 | 94.19 ± 2.91 | 0.00 | 0.00 | 0.00 | 94.15 ± 4.75 |
NAA/ZT | 0.00/0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.00/0.01 | 50.00 ± 4.10 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.00/0.10 | 37.55 ± 0.55 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.00/1.00 | 75.69 ± 1.69 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.00/10.00 | 100.00 | 7.12 ± 1.37 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.00/20.00 | 88.25 ± 052 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.01/0.00 | 36.42 ± 1.24 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.01/0.01 | 41.67 ± 0.68 | 0.00 | 0.00 | 0.00 | 2.93 ± 2.05 |
NAA/ZT | 0.01/0.10 | 60.65 ± 1.65 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.01/1.00 | 80.98 ± 1.89 | 8.68 ± 0.86 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.01/10.00 | 81.35 ± 1.35 | 9.47 ± 0.54 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.01/20.00 | 100.00 | 0.00 | 0.00 | 0.00 | 0.00 |
NAA/ZT | 0.10/0.00 | 31.36 ± 1.91 | 0.00 | 0.00 | 0.00 | 6.35 ± 1.32 |
NAA/ZT | 0.10/0.01 | 35.29 ± 1.08 | 0.00 | 0.00 | 0.00 | 14.38 ± 1.22 |
NAA/ZT | 0.10/0.10 | 54.37 ± 4.32 | 0.00 | 0.00 | 0.00 | 2.99 ± 0.85 |
NAA/ZT | 0.10/1.00 | 93.33 ± 5.20 | 0.00 | 3.00 ± 1.52 | 0.00 | 0.00 |
NAA/ZT | 0.10/10.00 | 93.89 ± 1.47 | 28.10 ± 1.28 | 0.00 | 7.38 ± 1.56 | 3.14 ± 2.56 |
NAA/ZT | 0.10/20.00 | 94.45 ± 7.20 | 27.31 ± 1.62 | 0.00 | 8.60 ± 1.28 | 0.00 |
NAA/ZT | 1.00/0.00 | 46.76 ± 4.18 | 0.00 | 0.00 | 0.00 | 46.79 ± 2.52 |
NAA/ZT | 1.00/0.01 | 96.87 ± 4.25 | 0.00 | 0.00 | 0.00 | 96.85 ± 2.20 |
NAA/ZT | 1.00/0.10 | 100.00 | 0.00 | 0.00 | 0.00 | 100.00 |
NAA/ZT | 1.00/1.00 | 60.78 ± 1.35 | 15.24 ± 1.63 | 0.00 | 2.58 ± 1.15 | 45.55 ± 3.25 |
NAA/ZT | 1.00/10.00 | 100.00 | 80.00 ± 4.58 | 0.00 | 18.51 ± 1.36 | 16.79 ± 1.54 |
---|---|---|---|---|---|---|
NAA/ZT | 1.00/20.00 | 83.96 ± 4.78 | 77.40 ± 1.65 | 0.00 | 20.11 ± 1.85 | 6.57 ± 2.06 |
NAA/ZT | 10.00/0.00 | 92.94 ± 1.25 | 0.00 | 0.00 | 0.00 | 92.98 ± 3.55 |
NAA/ZT | 10.00/0.01 | 100.00 | 0.00 | 0.00 | 0.00 | 100.00 |
NAA/ZT | 10.00/0.10 | 97.25 ± 4.25 | 0.00 | 0.00 | 0.00 | 97.02 ± 2.80 |
NAA/ZT | 10.00/1.00 | 68.84 ± 2.53 | 0.00 | 0.00 | 0.00 | 68.88 ± 4.51 |
NAA/ZT | 10.00/10.00 | 71.33 ± 6.35 | 22.60 ± 1.26 | 0.00 | 10.12 ± 1.65 | 48.45 ± 2.30 |
NAA/ZT | 10.00/20.00 | 83.45 ± 1.42 | 27.85 ± 3.45 | 0.00 | 15.23 ± 4.05 | 55.66 ± 3.31 |
NAA/ZT | 20.00/0.00 | 94.17 ± 5.32 | 0.00 | 5.85 ± 2.60 | 0.00 | 94.19 ± 2.56 |
NAA/ZT | 20.00/0.01 | 96.75 ± 2.69 | 0.00 | 0.00 | 0.00 | 96.76 ± 3.12 |
NAA/ZT | 20.00/0.10 | 100.00 | 0.00 | 0.00 | 0.00 | 100.00 |
NAA/ZT | 20.00/1.00 | 100.00 | 0.00 | 0.00 | 0.00 | 100.00 |
NAA/ZT | 20.00/10.00 | 100.00 | 0.00 | 0.00 | 0.00 | 84.35 ± 3.01 |
NAA/ZT | 20.00/20.00 | 60.74 ± 1.35 | 0.00 | 0.00 | 0.00 | 60.77 ± 2.34 |