Establishment of an in vitro Tissue Culture and Rapid Propagation System using Lonicera hypoglauca and Content Assessment of Total Flavonoids and Chlorogenic Acid

Lonicera hypoglauca is a traditional Chinese medicinal plant.In this study,the tender young leaves of L.hypoglauca were used for the first time as the explants to establish a rapid in vitro propagation and regeneration system.The results revealed that the optimal time for disinfection of the explants was 8 min and the optimal medium for callus induction was MS+2,4-D 4.0 mg·L^(-1)+sucrose 30 g·L^(-1),with an average callus induction rate of 86.67%.The optimal medium to induce differentiation of callus to bud was MS+6-BA 1.0 mg·L^(-1)+NAA 0.10 mg·L^(-1)+sucrose 30 g·L^(-1),with an average germination rate of 83.33%.The optimal medium to induce multiplication was MS+6-BA 1.5 mg·L^(-1)+NAA 0.05 mg·L^(-1)+sucrose 30 g·L^(-1),with a multiplication coefficient of 5.42.The optimal medium for root induction was 1/2 MS+NAA 0.15 mg·L^(-1)+activated carbon 0.3 g·L^(-1)+sucrose 15 g·L^(-1),with an average rooting rate of 91.11%.The survival rate of tissue-cultured seedlings in nutrient soil cultivation medium was as high as 100%.The total flavonoid content and chlorogenic acid content in the explant,callus tissue and regenerated plant were 1.83%,2.27%,1.33%and 2.77%,1.83%,1.74%respectively.This study provides novel insights into the rapid propagation and mass production of L.hypoglauca seedlings at an industrial scale and that it exhibits important application value and future prospects.

Rapid Propagation of Chirita ophiopogoides in Vitro

A method for in vitro culture and rapid propagation of Chirita ophio- pogoides was developed using leaves as explants in this study, The results indicat- ed that the medium MS＋6-BA 0.1 mg/L＋NAA 0.1 mg/L was suitable for bud induc- tion and seedling regeneration from leaves in primary culture. The media MS＋0.5 mg/L 6-BA＋0,1 mg/L NAA＋10% banana＋5% potato and MS＋0.5 mg/L 6-BA＋0.5 mg/L NAA＋2% banana were very suitable for callus multiplication and seedling hardening in subculture, and the proliferation coefficients were 7,9 and 5.6 per 60 d respec- tively. The optimal rooting medium was MS and the rooting rate was 100% on day 30 of culture. The rooted plantlets of C. ophiopogoides were transplanted in green- house with humus soil and 92.5% survived. Theoretically, using the rapid propaga- tion system, about 20 176 seedlings can be reproduced from a sterile plantlet in a year.

Embryo culture and rapid propagation of Syringa

Embryo of lilacs (Syringa L) culture in vitro and the rapid propagation were studied. The orthogonal experiments, in-cluding the selection of basal medium, embryo age and other factors such as sugar, benzyladenine (BA), naphthalene acetic acid (NAA) and glutamine (Gln), were carried out. The results indicated that the optimal medium for embryo culture was Monnier medium supplemented with NAA (0.001 mgL-1), BA (0.1 mgL-1), sugar (50 gL-1), and Gln (400 mgL-1), with a germination rate of 91.7% at least; the optimal embryo age was 50 d; and Gln had significant effects on the germination rate of embryo. Moreover, the optimal medium for subculture was MS+BA (2 mgL-1)+NAA (0.001 mgL-1)+Gln (0.5 mgL-1), with the propaga-tion coefficient of 3.6 at least.

Sterile Germination of the Hybrid Seeds of [Den. Burana Green Star × Den. Rainbow-compactum] and Rapid Propagation of F_1 Plantlets via Tissue Culture

[Objective] This study was to germinate the hybrid seeds of [Den.Burana Green Star × Den.Rainbow-compactum] under aseptic condition and to explore the parameters for rapid propagation of F1 plantlets via tissue culture.[Method]Hybridization between Den.Burana Green Star（female parent）and Den.Rainbow-compactum（male parent）was performed and the in vitro culture and proliferation of F1 hybrids were studied using eight different basic media including MS,1/2MS,1/3MS,1/4MS,B5,N6,modified Knudson and H.[Result]Improved Knudson medium appended with 1.0 mg/L 6-BA,1.0 mg/L NAA and 10% mature banana puree performed best in F1 seed germination under aseptic condition,as well as the rapid propagation of protocorm-like body.Of all the eight media tested,1/2MS is the medium most suitable for the in vitro rapid propagation of the F1 seedlings.Efficiency of eight media in the in vitro rapid propagation was in order 1/2 MS MS1/3 MS1/4 MS ≈N6 improved Knudson ≈B5H.NAA presented better rooting and growth-promoting effect in the in vitro rapid propagation of the F1 seedlings than IBA.And the optimal NAA concentration to recommend from our experiment results was 2.0 mg/L.[Conclusion]Our experimental results provided mature method and important technological information for hybrid breeding dendrobium.

Aseptic Seedling and Rapid Propagation of Ludisia discolor

[Objective] This study was conducted to establish a system for aseptic seeding and rapid propagation of a wild Chinese herbal medicine Ludisia discolor. [Method] The seeds of L. discolor were germinated in four different culture media MS, 1/2MS, Hyponex No.1 （3.0 g/L）, and 1/2 Hyponex No.1 （1.5 g/L）, and the ger- mination rate was calculated 80 d later to select the optimal medium for seed ger- mination of L. discolor. Then, by culturing the protocorms and buds of L. discolor in the media supplemented with different hormone combinations （6-BA and NAA） or organic substrates （potato juice, banana juice and coconut milk）, the optimal medi- um compositions for multiplication and .differentiation, seedling hardening and rooting of L. discolor were respectively determined. [Result] After 80 d of dark culture, 86.67% of the seeds inoculated into the medium 1/2 Hyponex No.1 germinated and developed into protocorms, proving 1/2 Hyponex No.1 was most suitable for seed germination of L. discolor among the four basic media, while MS medium had the worst effects. In culture medium of 1/2 Hyponex No.1＋2.0 mg/L 6-BA＋100 ml/L co- conut milk supplemented with 0.4 or 0.6 mg/L NAA, a mixed body of protocorms and small buds were induced after being cultured for 45 d, and the multiplication rate was 6.11, which was higher than that in the medium supplied with 0.2 or 0.8 mg/L NAA. By adding 10% potato juice, 10% banana juice or 100 ml/L coconut milk into Hyponex No.1＋1.0 mg/L NAA, it was found that 10% banana juice had better effects in hardening and rooting of L. discolor planlets than 10% potato juice and 100 ml/L coconut milk. The small buds grew into plantlets about 80 d later in the medium with banana juice, the rooting rate was 93.33%, and the plantlet height was 7.19 cm. After hardening-culture for 10 d in a greenhouse, the L. discolor plantlets were transplanted into the mixed matrix of peat soil and lava （at a ratio of 3：1）, and 91.7% of them survived 60 d later. [Conclusion] A relatively perfect propa- gation system of L. discolor was established by using aseptic seeds as explants in this study, which can be used for the protection of L. discolor germplasm resources, seedling propagation and industrial development.

Study on Virus-free Culture and Rapid Propagation Technology of Xiangshu Series Varieties of Sweet Potato

[Objective] Sweet potato virus disease had a significant harm to the yield and quality of sweet potato, directly causing the degradation of sweet potato vari- eties and even the harvest failure. Therefore, the detection and removal of sweet potato virus and the establishment of rapid propagation method of sweet potato is of great significance to ensure the stable inheritance of excellent characters of sweet potato, prevent the spread of sweet potato virus and develop sweet potato industry. [Method] With Xiangshu series varieties of sweet potato, Xiangshu 15 and Xiangshu 19 as the research materials, a virus-free culture program was established for meristem tip apex tissue culture of different cultivars, and a rapid propagation method was developed for virus-free seedlings. [Result｜ On the basis of analysis on seedling emergence rate, the optimal addition scheme of plant hormones in the MS culture medium of Xiangshu 15 was 6-BA 3.0 mg/L ＋ NAA 1.0 mg/L, and the opti- mal plant hormone addition scheme for Xiangshu 19 was 6-BA 2.0 mg/L ＋ NAA 0.67 mg/L Under the developed rapid propagation system, the annual reproductive coefficient was up to 49 152, far higher than that （20 000） in field. IConclusionl Based on the actual production, combined with the meristem tip apex tissue culture, a comprehensive prevention and control measure was put forward, which included virus detection, early warning, removal and virus-free seedlings breeding, tt was of great strategic significance to improve the yield and quality of high-quality sweet potato and ensure the healthy development of sweet potato industry in China.

Tissue Culture and Rapid Propagation of Spathiphyllum kochii Engl. et Krause

[Objectives] This study was conducted on tissue-culture rapid propagation techniques of Spathiphyllum kochii Engl. et Krause. [Methods] With lateral buds of S. kochii as explants, the effects of such four basic media as MS, B5, Nitsch and Wpm and the ratio of two hormones(1, 2, 3 mg/L 6-BA and 0.1, 0.3, 0.5 mg/L NAA) on bud proliferation of S. kochii were studied by the complete test method, and the effect of the ratio of the two hormones(0.25, 0.50, 1.00 mg/L NAA and 1.0, 1.5 mg/L IBA) on rooting of S. kochii as well as the effect of substrate ratio(perlite and peat soil at 1∶9, 2∶8, 3∶7, 4∶6 and 5∶5) on its transplanting survival rate were also studied. [Results] The best basic medium for the rapid propagation of S. kochii was MS. The best hormone ratio suitable for bud proliferation was 2 mg/L6-BA+0.1 mg/L NAA, and the average number of buds proliferated at 45 d was 3.04. The average bud height was 2.05 cm; the most suitable medium for rooting was 1/2 MS+1 mg/L IBA+0.25 mg/L NAA, and its rooting rate was 100%; and the best transplanting substrate was 5∶5 perlite and peat. The soil ratio had a survival rate of 94%. [Conclusions] This study provides a theoretical basis for the improvement of the transplanting survival rate of test-tube S. kochii plantlets.

Effect of Different Plant Growth Regulators on Callus Induction and in vitro Rapid Propagation of Wild Petunia Juss.

In this study,the seeds of wild Petunia Juss.were used as explants to investigate the optimal condition for tissue culture.Several different kinds and concentrations of growth regulators were adopted to produce more multiple bud clumps,callus or roots in this study.The experiments may provide experimental foundation for the rapid propagation technology and establishment of tissue culture system for wild Petunia Juss.

Tissue Culture Rapid Propagation and Transplantation Techniques of Anoectochilus roxburghii (Wall) Lind.

[Objectives]To screen the tissue culture rapid propagation formula suitable for each tissue culture stage of Anoectochilus roxburghii( Wall) Lind. [Methods] The stem segments of Fujian A. roxburghii were used as explant to study the tissue culture rapid propagation and transplantation techniques. The comparative experiment was carried out to study the effects of different hormone concentrations on the induction of stem segments,proliferation of cluster buds,rooting and seedling hardening of A. roxburghii,and study the effects of transplantation matrix on the transplantation of A. roxburghii. [Results]MS + 0. 5 mg/L NAA + 2 mg/L BA + 20 g/L sucrose + 6 g/L agar was suitable for induction of stem segments of A. roxburghii; MS + 0. 5 mg/L NAA + 2 mg/L BA + 1 mg/L KT + 25 g/L sucrose + 6 g/L agar was most suitable for proliferation of cluster buds of A. roxburghii; MS + 1. 0 mg/L IBA + 1. 0 mg/L NAA + 1 g/L activated carbon + 50 g/L mashed banana + 25 g/L sucrose + 6 g/L agar was most suitable for rooting and seedling hardening of A. roxburghii; using peat soil: fine sand( 3∶ 1) as transplantation matrix,the survival rate was the highest. [Conclusions] The experiment results are expected to provide references for factory production of A. roxburghii.

Tissue Culture and Rapid Propagation of Pedicels of Hemerocallis hybrida

[ Objective] This study aimed to develop tissue culture and rapid propagation methods for pedicels of HemerocaUis hybrida. [ Method] Tender pedicels of dwarf H. hybrida were used as experimental materials to sdect callus induction, subculture and rooting media for rapid propagation of H. hybrida.[ Result ] MS ＋ 2.0 - 3.0 mg/L 6-BA ＋ 0.2 - 0.3 mg/L NAA, MS ＋ 1.0 - 2.0 mg/L 6-BA ＋ 0.1 - 0.2 mg/L NAA, MS and 1/2MS ＋ 0.2 mg/L NAA were the appropriate me- dium for callus induction, subculture and rooting, respectively. [ Conclusion] The in vitro culture and clustered seedling rooting technology used in this study are effective methods for rapid propagation of H. hybrida, which provide technieal reference for industrialized production of H. hybrida.

Sterile Germination of Dendrobium chrysotoxum Seeds and Rapid Propagation of Its Plantlets

[Objective] This study was to produce plant seeds on a large scale via sterile germination of the capsules of Dendrobium chrysotoxum and rapid propagation technique.[Method] A large amount of protocorm-like bodies produced from the sterile germination of D.chrysotoxum capsules,were transferred to four different kinds of bud induction media to obtain the optimal media suitable for plantlet differentiation and growth;and then with N6 as basic medium,1.0,1.5 and 2.0 mg/L of NAA and IAA were tested to obtain the optimal media suitable for rooting.[Results] On the medium of MS appended with 1 mg/L 6-BA ＋10% banana puree ＋ 20 g/L sucrose ＋6 g/L agar＋1 g/L AC,seed germination rate was up to 90%.The optimal medium for differentiation of D.chrysotoxum protocorm-like bodies was N6＋ 2 mg/L NAA ＋ 0.5 mg/L 6-BA ＋10% banana puree ＋ 20 g/L sucrose ＋ 0.5 g/L peptone ＋ 5.8 g/L agar ＋0.5 g/L AC,grown from which the plantlets were even and orderly in height;and the optimal medium for rooting was N6＋1.5 mg/L NAA ＋10% banana puree ＋ 20 g/L sucrose ＋ 5.8 g/L agar ＋1 g/L AC,grown from which the plantlets developed more,robust and orderly roots,and their leaves were in dark green color.[Conclusion] Our results provided reference for the rapid propagation of D.chrysotoxum.

Establishment of a Rapid Propagation Protocol of GLRaV-3,GFKV,and GRSPa Free Grapevine Rootstocks

Nursery plant propagation by grafting has been widely used in modern viticulture to minimize the damage caused by biotic and abiotic stresses.In grapevine(Vitis spp.),an effective way to control disease damage is to provide producers and growers with pathogen-free stock plants.In this study,five grapevine rootstock varieties,‘SO4’,‘101-14’,‘5BB’,‘110R’and‘1103P’,were selected as explants to establish an in vitro culture protocol,and three species of grapevine viruses were tested by real-time RT-PCR.The results showed that MS medium supplemented with 0.2 mg/L IBA,1.0 mg/L 6-BA,0.5 mg/L KT,4.0 mg/L adenine for culture initiation,and WPM supplemented with 0.2 mg/L IBA for subculture were suitable for all five rootstock varieties,with multiplication coefficients ranging from 1.6 to 4.4.Virus testing showed that single RT-PCR was more effective for detecting the three viruses compared to double or triple RT-PCR.Only plantlets free from the aforementioned viruses were retained for subculture.Plantlets were hardened at room temperature under natural lighting in Hoagland solution for 2 weeks and transplanted to pots filled with mixed media in a greenhouse.This protocol is applicable for rapid propagation of the five grapevine rootstock varieties and can be used for commercial production of virus-free grapevine stocks.

Rapid Propagation of Virus-free Sugarcane Plantlets via Temporary Immersion Bioreactor System

By employing temporary immersion bioreactor system(TIBs),we studied virus-free culture of seedlings from sugarcane varieties ROC16 and ROC22,from medium recipe,inoculation amount,sucrose concentration,and variety difference. The results showed,using this method,that proliferation rate of ROC16 improved by 40 times,per flask generated about 800 plantlets; of ROC22 improved by 30 times,per flask generated about 400-600 plantlets. The results provided basis for using TIBs in rapid propagation of plantlets via tissue culture.

Study on the rapid propagation of Phalaenopsis hybrid

This paper studied the rapid propagation technology of Phalaenopsis hybrid by using the peduncle buds as the explants, and screened out a kind of culture medium, which was simple, rapid, available and high rate of propagation.

Cutting Rapid Propagation Technology for Antigonon leptopus in Tropical Areas

The shoots with good functional leaves were selected, from which cutting slips with the length of about 20 cm were cut, and 2 nodes were left for the ones with long sections. All leaves could be kept or 1/3 of each leaf could be cut. The cutting depth was that making the bottom node touch the substrate, and the cutting slips with short nodes should have 1/3 of the length insert in the soil. Each pot was planted with 2 - 3 cutting slips, and the substrate around the cutting slips was pressed by hands, followed by enough watering. And then white plastic bags were used to cover the pots, which were then placed in the place with the shading rate of 75%. The method had low costs but good landscape effect, promising with good development prospects, which could provide references for the promotion of out- of-season rapid propagation technology of Antigonon leptopus in tropical areas.

Rapid Propagation of Rhynchostylis retusa in Vitro

An efficient regeneration system of Rhynchostylis retusa was established to provide technical reference for the application of tissue culture tube seedlings in production.The mixtures of callus and protocorm from aseptic germination were used as explants.The optimal media of each stage was selected for callus proliferation,protocorm occurrence and growth,rejuvenation and rooting via a single,complete combination and orthogonal experiment.The results showed that the optimal medium for callus proliferation,protocorms occurrence and growth was 1/2 Murashige and Skoog(MS)medium adding 50 g·L^(−1) banana puree,0.1 mg·L^(−1)α-naphthaleneacetic acid(NAA),1.5 mg·L^(−1)6-benzylaminopurine(6-BA)and 1.0 mg·L^(−1) kinetin(KT)with 17.33 proliferation coefficient of callus and 19.63 occurrence coefficient of buds after 90 days.Then the buds occurred from protocorm were cultured on 1/2 MS medium including 100 g·L^(−1) banana puree,1.0 mg·L^(−1) NAA,2.0 mg·L^(−1)6-BA and 0.05 mg·L^(−1) KT,in which the proliferation coefficient of callus was 10.32 and occurrence coefficient of buds reached 17.87.In the further subculture,the same medium was simultaneously used for callus proliferation,protocorm occurrence and bud growth.The plantlets developed roots in 1/2 MS medium containing 70 mL·L^(−1) coconut water and 1.5 mg·L^(−1) NAA with 100%rooting rates after 90 days.The survival rate was more than 90%after domestication and transplantation.This regeneration protocol will provide technique foundation for protecting wild resource and developing artificial cultivation.

A Study on Tissue Culture and Rapid Propagation ofGinger

The buds of ginger grown in Tongdao Dong Autonomous County were used as explants, the tissue culture and rapid propagation of ginger were studied and the CMV (Cucumber mosaic virus) and TMV (Tobacco mosaic virus) of ginger tissue culture seedlings were detected. The results showed that MS+6-BA 4.0 mg/L+NAA 0.2 mg/L was the suitable medium for the proliferation induction and differentiation of ginger seedlings. This medium can realize the synchronous culture of ginger seedling proliferation and rooting, and one-step seedling re fining and transplanting, and the proliferation multiple can reach 3.00. The suitable rooting medium was 1/2MS+0.4 mg/L NAA. The survival rate of coir and vermiculite was the highest among the 4 culture mediums, reaching 100%. The survival rate of peat and cottonseed shell was very low, among which the ginger seedling transplanted with vermiculite grew more robustly than that transplanted with coir, with developed root system, more adventitious roots and less yellowing of leaves. No CMV and TMV were found in the tissue culture seedlings of ginger. The tissue culture seedlings can be used for the production of non-toxic ginger seedlings.

Research on Rapid Propagation of Gongshui Pomelo by Tissue Culture

[Objective] This study aimed to investigate the optimal medium and hor-mone combinations for efficient rapid propagation of Gongshui pomelo and analyze key technical measures in the tissue culture process. [Method] Stem tips and stem segments with buds were col ected from four varieties of pomelo adult trees as explants, to investigate the main effect and key regulatory factors of vegetative organs and tissue culture explants and to propose a series of measures to prevent and control microbial contamination. Final y, an efficient rapid propagation technology system of Gongshui pomelo was established. [Result] Spring shoot explants contained large amounts of auxin, cytokinins, gibberel ins and other growth regulators, which could be used for tissue culture with high bud generation rate and rapid growth. Different conditions led to various culture results. Specifical y, mature pomelo seeds should be generated on semisolid 1/2MS medium and transferred to solid MS medium for incubation. The propagation coefficient of stem segments with axillary buds was greater than that of stem tips, exhibiting significant differences. In ad-dition, the optimal hormone combination was 6-BA 0.5 mg/L ＋ NAA 0.5 mg/L, which significantly promoted the induction and differentiation of adventitious buds. [Conclusion] This study provided basis for basic research, production and application of pomelo germplasm resources.

Study on Tissue Culture and Rapid Propagation of Tilia amurensis

To explore the establishment of a tissue culture and rapid propagation system of Tilia amurensis,the effects of basic medium and concentrations and ratios of plant growth regulators on tissue culture and rapid propagation of T.amurensis were studied.The results showed that 1/2 MS medium was the most suitable proliferation medium,and the proliferation coefficient could reach 13.5 after adding 0.05 mg/L 6-BA and 0.03 mg/L IBA;MS medium was the most suitable medium for strong plantlets and rooting,and the best medium for strong plantlets was MS+0.1 mg/L 6-BA+0.1 mg/L IBA+0.03 mg/L GA_(3),with which the average plantlet height reached 5.15 cm;and the best rooting medium was MS+1.0 mg/L6-BA+0.05 mg/L NAA,with which the rooting rate was 93.3%and the number of roots was 5.7 roots.

Overview of Research on Tissue Culture and Rapid Propagation Technology of Pholidota

The genus Pholidota has good medicinal value,and is often over-excavated by humans.Coupled with its low natural reproduction rate,Pholidota is almost endangered.This paper summarized the tissue culture and rapid propagation technology of Pholidota in recent years,aiming to provide key technical support for resource protection and development of Pholidota and preliminary foundation and technical support for follow-up related research.