An Efficient Plant Regeneration System of Hydrangea bretschneideri Dipp via Stem Segments as Explants

Hydrangea bretschneideri Dipp is a highly popular ornamental plant for garden decoration.Genetic engineering technology has been successfully used in many plant species,but it is limited in Hydrangea.Here we established an efficient regeneration system by using stem segments as explants for the first time.In our study,the plant growth regulators(PGRs)were evaluated at the different regeneration processes,including axillary shoots regeneration and root induction.We found that the optimal concentration for axillary buds’induction was 2.0 mgL^(-1)6-BA and 0.5 mgL^(-1)1 IAA,its highest induction rate was 70%.Moreover,the highest axillary shoots proliferation coefficient was 10.7 on the Murashige and Skoog(MS)medium with 2.0 mgL^(-1)6-benzyladenine(BA),0.2 mgL^(-1)indole-3-butyric acid(IBA),and 1.0 mgL^(-1)gibberellin A3(GA3).The highest frequency of root induction was 80.0±0.06%by culturing the elongated shoots in 1/2 MS medium containing 0.1 mgL^(-1)IBA.In summary,our study will provide an effective technology for large-scale propagation and important pathway for promoting the popularization and application of Hydrangea bretschneideri Dipp.

Establishment of Tissue Culture Regeneration Systems of Stem Segments of Euphorbia tirucalli

[Objective] The aim was to study the conditions of tissue culture regeneration seedling by using the stem segments of Euphorbia tirucalli and determine the optimum culture condition of each culture stage,so as to provide references for the factory production and relative study of tissue culture seedling of E. tirucalli. [Method] Taking the stem segments of E. tirucalli as explants,the effects of various mediums on germination rate,multiplication coefficient and rooting rate were studied. [Result] The optimum induction medium of germination culture was 1/2MS＋NAA 0.02 mg/L＋6-BA 1.0 mg/L,with differentiation rate of 89.7%; the best subculture medium was 1/2MS＋NAA 0.02 mg/L＋6-BA 0.60 mg/L＋AD 3.0 mg/L,with multiplication coefficient of 5.70; the optimum rooting culture medium was 1/2MS＋NAA 0.40 mg/L＋IBA 0.4 mg/L,with rooting rate of 100% and transplanting survival rate of 80%. [Conclusion] The tissue culture conditions of stem segments of E. tirucalli were determined primarily.

Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

Establishment of Regeneration System of Bougainvillea ‘Yunnan Purple’ Stem Segment

To optimize the regeneration system of Bougainvillea, the effects of different hormone ratios and concentrations on axillary bud induction, proliferation and re-rooting were studied using annual semi-lignified branch cuttings of Bougainvillea ‘Yunnan Purple’ as experimental materials. The results showed that MS+6-BA 2.5 mg/L+NAA 0.1 mg/L was the optimal medium for stem axillary bud initiation, and the initiation rate reached 91.3%. The optimal medium for axillary bud proliferation was MS+6-BA 1.0 mg/L+NAA 0.2 mg/L, and the proliferation coefficient was 3.28. The optimal rooting medium was 1/2 MS+IBA 1.0 mg/L, with the rooting rate of 90% and the average root number of 7.4. After 15 d of hardening seedling, the survival rate of the sterile seedlings was 97.83%. This study laid a basis for rapid propagation and genetic transformation system of Bougainvillea.

In vitro Plant Regeneration from the Mature Tissue of Navel Orange (Citrus sinensis L. Osbeck) by Direct Organogenesis

An efficient in vitro regeneration system by direct organogenesis from mature nodal and internodal stem segments ofNewhall navel orange (Citrus sinensis L. Osbeck) was developed. Illuminating conditions together with plant growthregulators affected the adventitious bud regeneration frequency and efficiency. The initial 15 d darkness inoculation isbeneficial for the adventitious bud regeneration. The highest regeneration frequency (85.2%) and bud formationefficiency (3.7 per responsive internodal stem segment) were obtained in the media supplemented with 1.0 mg L-1 BAPand 0.5 mg L-1 NAA. ABA at 0.2 mg L-1 positively affected the bud formation efficiency, which amounted to 8.5 buds perinternodal segment in the presence of BAP at 1.0 mg L-1. The adventitious shoots successfully rooted and weretransferred to the soil.

Investigation on the Morphological Characteristics of Dendrobium officinale Plantlets Propagated from Different Explants

[ Objective] This study aimed to investigate the differences in morphological characteristics of Dendrobium officinale plantlets propagated from different explants. [ Method] Randomly 1 000 D. offtcinale plantlets propagated via different regeneration pathways were selected for morphological investigation and classification. [ Result] D. officinale plantlets propagated from stem segment explants exhibited highly consistent morphological characteristics, while those propagated from seed explants exhibited a variety of morphological characteristics. [ Conclusion] Therefore, using seed explants for regeneration can effectively broaden the germplasms resources of D. officinale.

Friable Callus Induction of Hedera nepalensis var.sinensis

[Objectives]The purpose was to establish an induction system for friable callus of Hedera nepalensis var.sinensis with different parts.[Methods]By screening the most suitable explant and adjusting the hormone ratio of medium,friable calli of H.nepalensis var.sinensis were induced.[Results]The calli could be induced from leaves,petioles and stem segments,but the ideal explant was stem segments,with induction rate reaching 98%.The optimal medium for callus proliferation was MS+0.5 mg/L KT+1.0 mg/L 2,4-D+30.0 g/L sucrose.After 3-4 generations of subculture on MS+0.5 mg/L BA+1.0 mg/L 2,4-D+30.0 g/L sucrose,favorable friable calli of H.nepalensis var.sinensis were obtained.[Conclusions]The friable calli induced in this experiment can lay a foundation for in-vitro regeneration and cellular secondary metabolite production of H.nepalensis var.sinensis.