| ObjectiveAnoectochilus roxburghii (Orchidaceae) is a rare perennial, medicinal herb, which has the effect of clearing away a toxic fever, cooling blood, and dehumidification detoxification. A. roxburghii can cure hemoptysis of pulmonary tuberculosis, diabetes, nephritis, cystitis, myasthenia gravis, nocturnal emission, rheumatic and rheumatoid arthritis, infantile convulsion, women leucorrhea and snake bite. A. roxburghii is not only valued in traditional medicine but also prized as an ornamental plant because of its attractive, golden-yellow-veined foliage. A. roxburghii occupies an extremely narrow habitat in its native range, and is very sensitive to the surrounding environment. Natural populations of A. roxburghii are becoming increasingly rare because of continued habitat destruction, and natural propagation of A. roxburghii is very difficult. To ensure the sustainability of this useful resource, artificial cultivation techniques based on plant tissue culture have been used to establish a rapid propagation system for A. roxburghii. So, in this study, the rare earth elements (La and Ce) have been applied to in vitro cultured A. roxburghii. And we have studied the effect of REEs on in vitro cultured A. roxburghii by adding different concentrations of La (NO) 3 and Ce (NO) 3 to improve and perfect the in vitro regeneration of A. roxburghii, and we can obtain regenerated A. roxburghii with good quality and shorten its cultured period. In order to reveal the possible mechanism, we have also studied the effect of La(NO3)3 and Ce(NO3)3 on chlorophyll contents, root activity, NR, SOD, POD and CAT enzyme activities, MDA contents and other physiological indicators. At the same time, the content of effective chemical components like flavonoids and gastrodin were also investigated.MethodsIn this research, the in vitro propagation system of A. roxburghii was established, and the effect and possible mechanism of REEs (La and Ce) on the growth of in vitro cultured A. roxburghii were investigated by adding different concentration of La(N03)3 and Ce(N03)3. The following were the details:1.Establishing the seed germination system of wild A. roxburghii, and investigating the effect of various culture media (MSn 1/2MS and MS), different concentrations, different kinds of organic additives and plant hormones, different explant materials on the growth of in vitro cultured A. roxburghii. By those steps, the favorable rapid propagation system for A. roxburghii was developed.2. Investigating the effect of REEs on growth of in vitro cultured A. roxburghii by adding different concentrations of La(NO3)3 and Ce(NO3)3 into the culture media and transplanting experiment;3. By calculating content of physiological indicators such as chlorophyll, root activity, NR, SOD, POD and CAT enzyme activities, as well as MDA of regenerated A. roxburghii in order to preliminarily investigate the possible mechanism;4. By calculating the content of effective chemical compositions like total flavonoids and gastrodin, we can find some clues to explain the possible mechanism on promoting growth of in vitro cultured A. roxburghii with REEs.Results1. Establishment of in vitro propagation system of A. roxburghiiMature wild-seeds of A. roxburghii as explant materials were inoculated onto 1/2MS medium, the seeds absorbed water and were swollen, with embryos enlarged and testae beginning to rupture, then they became yellow-white protocorms after 30days culture. With the prolongation of the culture time, the original protocorms were growing, developing, proliferating and differentiating, and almost all protocorms became into green seedlings with 1-2 leaflets after 90days culture. Our results showed that the growth of A. roxburghii in the 1/2MS basic medium was better than that in the MS and modified MS basic medium. And it also showed that 100 g/L of potato extract,50 g/L of banana extract, and 15 g/L of potato powder could promote rapid growth of A. roxburghii apical buds. Additionally, A. roxburghii regenerated seedlings showed good growth as well as large leaves and a few lateral buds by adding 1.0 mg/L 6-BA or 1.5 mg/L NAA. Moreover, the optimum concentration of TDZ for the growth of mid-stem segments 2.0 mg/L, while 4.0 mg/L during the early culture period (3-4 months) and reduced to 2.0 mg/L during the latter culture period (4 months later) for the growth of basal rhizome segments. Furthermore, the optimum ratio of hormones promoting callus formation from stem segment were 1.0 mg/L 6-BA+O.5 mg/L NAA+0.5 mg/L ZT+0.2 mg/L 2,4-D, which provided the highest percentage callus response of 90.7%; while The optimum ratio of hormones promoting callus formation from leaves were 1.0 mg/L 6-BA+0.5 mg/L NAA+0.2 mg/L ZT+0.2 mg/L 2,4-D, which provided the highest percentage callus response of 90.0%.2. Research about effect of REEs on growth of in vitro cultured A. roxburghiiThe results showed that the REEs (La and Ce) could effectively accelerate the plantlet growth of in vitro cultured A. roxburghii. Within a certain range of concentration, REEs first stimulated the plantlet growth as concentrations of La(N03)3 and Ce(N03)3 increased, and then inhibited plantlet growth once the contrations of La(N03)3 and Ce(N03)3were out of range. Adding 2.0 mg/L La(NO3)3 or Ce(N03)3 was more appropriate for the in vitro growth of early germinated seedlings from A. roxburghii seeds, which made the average height and average plant fresh weight reach the maximum. After 100 days of culturing, average heights of plantlets derived from apical buds were 6.0 cm with 5.0 mg/L La(N03)3 and 6.2 cm with 2.0 mg/L Ce(N03)3, which respectively increased by 28.0% and 32.0% as compared with that in the non-treated control group. The optimum concentration for shoot induction from mid-stem segments was 1.0 mg/L Ce(N03)3 which had a better proliferation times of 1.5-fold and an average length of 3.0 cm compared with 1.0-fold and 2.2 cm in the control group. Optimum growth from basal rhizome segments was achieved on media supplemented with 3.0 mg/L Ce(N03)3, which provided a better proliferation times of 5.1-fold and an average shoot length of 4.5 cm compared with corresponding control values of 2.0-fold and 3.5 cm.3. Research about effect of REEs on physiological indicators contents of regenerated A. roxburghiiThe study showed that a certain concentration of La(NO3)3 and Ce(NO3)3 can increase physiology indicators contents of in vitro cultured A. roxburghii. Within the concentration range of 0-5.0 mg/L La(N03)3 and Ce(NO3)3, some physiological indicators contents first increased with 0-2.0 mg/L and then decreased with 2.0-5.0 mg/L. And when the concentrations of La (NO3)3 and Ce (NO3) 3 both were 2.0 mg/L, the highest chlorophyll contents of A. roxburghii respectively increased by 38.5% and 44.2% compared with that in non-REE solution; the root activities were respetively 2.88-fold and 3.07-fold of that in control group; the NR activities reached highest and were respetively increased by 19.9% and 50.7% compared with that in control grou; the highest SOD activities were respetively 25.13 unit/mgprot and 23.88 unit/mgprot, which were respetively increased by 31.4% and 25.1% compared with that in control group; the highest CAT activities were respetively 13.73 unit/mgprot and 17.07 unit/mgprot, which were respetively 1.83-fold and 2.28-fold of that in control group; the highest POD activities were respetively 21.22 unit/mgprot and 32.89 unit/mgprot, which were respetively 1.52-fold and 2.35-fold of that in control group. By adding 0-5.0 mg/L La(N03)3 and Ce(NO3)3, MDA contents first decreased with 0-2.0 mg/L and then increased with 2.0-5.0 mg/L. And when the concentrations of La(NO3)3 and Ce(NO3)3 both were 2.0 mg/L, MDA contents were respetively 2.84 nmol/mgprot and 2.99 nmol/mgprot, while it was 3.48 nmol/mgprot in control group.4. Research about effect of REEs on effective chemical ingredients contents of regenerated A. roxburghiiLa(NO3)3 and Ce(NO3)3 at concentrations ranging from 0-5.0 mg/L can increase the total flavonoids contents of in vitro cultured A. roxburghii. Within the concentration range of 0-5.0 mg/L La(NO3)3 and Ce(NO3)3, the total flavonoids contents first increased with 0-2.0 mg/L and then decreased with 2.0-5.0 mg/L. And the appropriate concentration of La(NO3)3 and Ce(NO3)3 were both 2.0 mg/L, the highest total flavonoids contents of A. roxburghii were respectively 8.15 mg/g and 10.48 mg/g, which were respectively increased by 37.4% and 76.7% compared with that in non-REE solution. Thus, the changing chlorophyll contents, enzymes activity (NR, SOD, CAT and POD) and MDA contents in plant seedlings may be significant in explaining the possible regulatory mechanism of La(NO3)3 and Ce(NO3)3.ConclusionA. roxburghii germination system was established by using mature-wild seeds. Through using various basic culture media (MSã€1/2MS and modified MS), different concentrations, different kinds of organic additives and plant hormones, and different explant materials, the most appropriate culture condition was found to obtain good quality seedlings. And at the same time, the cultivation period was shorten and production costs was reduced. Meanwhile it can help to solve the resource shortage problem of A. roxburghii, provide technical support for the A. roxburghii factory production, and lay a foundation for the sustainable development of its resources. Certain concentration of La(N03)3 and Ce(NO3)3 can promote seedlings growth of A. roxburghii. In this study, through applying different concentration of La(NO3)3 and Ce(NO3)3, the external indicators such as plant height, leaf amount, node amount and fresh weight, together with internal indicators such as chlorophyll contents, root activity, enzymes activity (NR, SOD, CAT and POD), MDA contents and the active chemical components of total flavonoids, gastrodin were investigated. Thus, the changing chlorophyll contents, enzymes activity (NR, SOD, CAT and POD) and MDA contents in plant seedlings may be significant in explaining the possible regulatory mechanism of La(N03)3 and Ce(NO3)3. However, the clear mechanism still needs further study. |
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