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.

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.

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.

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.

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.

Primary Establishment of the Tissue Culture Technique and Regeneration System for Ornamental Lupinns polyphyllus

The purpose of this paper is to develop a system for tissue culture and rapid propagation of two ornamental lupins, Minaretie and Russell Prize. In view of screening out the better explant regeneration and suitable culture medium, through adding hormone 6-BA, NAA and 2, 4-D into MS and B5 basic culture medium, a series of experiments were carried out with the shoot tips, leaves, leaf petioles and stems from the asepsis seedling. The results showed that the shoot tips had favorableness on the rapidly propagation; MS＋6-BA 0.5 mg. L-1 for first generation, the induction rate of Minaretie and Russell Prize was 90.5% and 95.86% respectivdy; Minaretie had the highest propagation index （6.35） on MS＋6-BA 0.5 mg.L^-1＋NAA 0 mg-L^-1＋GA 30.8 mg. L^1＋AC 2 g. L^-1, but Russell Prize had the highest propagation index （7.24） on MS＋6-BA 0.5 mg.L^-1＋NAA 0.15 mg.L^-1＋GA3 1.0 mg.L^-1＋AC 0.5 g.L^-1; 1/2 MS＋NAA 0.25 mg.L^-1 was the best rooting medium. The ratios of getting roots of Minaretie and Russell Prize were 94.78% and 96.32%, respectively.

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.

In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants

Objective:To develop the reproducible in vitro propagation protocols for the medicinally important plants viz.,Achyranthes aspera(A.aspera)L.and Achyranthes bidentata(A.bidentata)Blume using nodal segments as explants.Methods:Young shoots of A.aspera and A.bidentata were harvested and washed with running tap water and treated with 0.1%bavistin and rinsed twice with distilled water.Then the explants were surface sterilized with 0.1%(w/v)HgCl_2 solutions for I min.After rinsing with sterile distilled water for 3-4 times,nodal segments were cut into smaller segments(1 cm)and used as the explants.The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog(MS)medium supplemented with 3%sucrose,0.6%(w/v)agar(HiMedia,Mumbai)and different concentration and combination of 6-benzyl amino purine(BAP),kinetin(Kin),naphthalene acetic acid(NAA)and indole acetic acid(IAA)for direct regeneration.Results:Adventitious proliferation was obtained from A.aspera and A.bidentata nodal segments inoculated on MS basal medium with 3%sucrose and augmented with BAP and Kin with varied frequency.MS medium augmented with 3.0 mg/L of BAP showed the highest percentage(93.60±0.71)of shootlets formation for A.aspera and(94.70±0.53)percentages for A.bidentata.Maximum number of shoots/explants(10.60±0.36)for A.aspera and(9.50±0.56)for A.bidentata was observed in MS medium fortified with 5.0 mg/L of BAP.For A.aspera,maximum mean length(5.50±0.34)of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A.bidentata(5.40±0.61)was observed in the very same concentration.The highest percentage,maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of 1BA.Seventy percentages of plants were successfully established in polycups.Sixty eight percentages of plants were well established in the green house condition.Sixty five percentages of plants were established in the field.Conclusions:The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A.aspera and A.bidentata.The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can he easily adopted for commercial large scale cultivation.

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.

Study on Application Technology for Tissue Culture and Propagation of Tagetes patula L.

[ Objective] This study aimed to investigate the tissue culture and propagation technology in Tagetes patu/a L. [ Method] By using tissue culture tech- nology, different mass fractions of 6-BA and NAA were added to MS medium to compare the effect of different culture medium on the rapid propagation of T. patu/a L. [Result] Shoot tips or stem segments of T. patu/a L. were used as explants for tissue culture with an appropriate sterilization time of 8 min; differentiation effect of shoot tips was better than that of stem segments; callus generation rate was high with the high content of growth regulators; MS medium containing O. 1 mg/L NAA and 1.5 rag/L 6-BA was used for subculture proliferation with a subculture period of 4 weeks; rooting rate of plantlet was the maximum （97%） in 1/2MS medium containing 0.2 mg/L NAA, and the root system was relatively developed. [ Conclusion] This study provided technical support for the industrialized seedling breeding of T. patula L.

Study on Virus-free and Rapid Propagation Technology of Artemisia selengensis sp.in Yangxin County

[Objectives]This study was conducted to culture virus-free tissue culture plantlets of mid-maturing green-stalk Artemisia selengensis sp.varieties in Yangxin County.[Methods]With bud tips of A.selengensis in Yangxin as explants and MS as the basal medium,screening and proportioning of plant growth regulators were performed to establish a virus-free and rapid propagation system for mid-maturing green-stalk varieties of A.selengensis in Yangxin.[Results]The optimal callus induction medium,adventitious shoot differentiation medium,adventitious shoot elongation medium and rooting medium for explants were MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+sucrose 25 g/L+agar 7 g/L,MS+6-BA 0.1 mg/L+NAA 0.1 mg/L+sucrose 25 g/L+agar 7 g/L,MS+6-BA 0.02 mg/L+NAA 0.02 mg/L+sucrose 25 g/L+agar 7 g/L and 1/2 MS+NAA 0.05 mg/L+sucrose 25 g/L+agar 7 g/L,respectively,and the seedling rate reached more than 95%.The culture conditions were as follows:temperature(25±2)℃,relative humidity 85%,illumination intensity 3000 lx,and illumination time 14 h/d.[Conclusions]This study has important practical significance for the purification and rejuvenation and large-scale industrial breeding of Yangxin A.selengensis 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.

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.

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.

Micropropagation of yellow mangosteen:a valuable endemic tree of India

We developed a method for in vitro regenera- tion of Garcinia xanthochymus （yellow mangosteen） from matured seed segments. Multiple shoots were induced on woody plant （WP） medium supplemented with cytokinins. An average of 11 shoots per explant were regenerated from mature seed segments on WP medium containing 20 μM 6-benzylaminopurine. Histological analysis revealed that hypodermal cells of seed segments were initially involved in active division, which later developed into meriste- moids, subsequently leading to the formation of shoot buds. Shoot elongation was achieved by repeated subculturing of seed explants in shoot regeneration medium. Rooting of shoots was achieved on WP medium supplemented with indole-3-butyric acid or s-naphthalene acetic acid. Plant- lets were transplanted to pots containing soil： compost （1：1） and survival rate was 90 %.

Effect of Vitamins on <i>In Vitro</i>Organogenesis of Plant

Vitamins are necessary compounds synthesized and utilized in plants. In tissue culture media, vitamin addition is not always common;since the amount needed by plants is relatively unknown and varies. Vitamins, in combination with other media constituents, have been shown to have direct and indirect effects on callus growth, somatic growth, rooting, and embryonic development. For example, different studies have shown that thiamine is associated with cytokinin and has a role in inducing callus growth and rooting. Moreover, thiamine was essential in facilitating the production of more secondary metabolites such as proteases in pineapple. Both biotin and riboflavin play a role in callus development as well. Specifically, riboflavin exerts different effects on plant rooting either positively and negatively. Vitamin D known to cause uptake of calcium in animal tissue, exerts a similar effect in plants. In addition, vitamin D causes cell elongation and meristematic cell division. Vitamin C, known for its anti-oxidative properties, has also enhanced shoot growth and rooting.

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.

珠芽魔芋组织诱导快速繁殖技术研究

以珠芽魔芋球茎为外植体,开展组织诱导快速繁殖技术研究,以期为珠芽魔芋的规模化种植提供技术支撑。结果表明:最佳萌发培养基为MS+0.5 mg/L 6-BA+0.1 mg/L 2,4-D+30 g/L蔗糖+5 g/L琼脂,萌发率达100%;最佳愈伤组织诱导培养基为MS+2.0 mg/L 6-BA+0.5 mg/L GA3+30 g/L蔗糖+5 g/L琼脂,叶片和叶柄形成愈伤组织的诱导率分别为99.17%、92.50%;最佳不定芽诱导培养基为MS+1.0 mg/L 6-BA+0.4 mg/L NAA+25 g/L蔗糖+5 g/L琼脂,叶片和叶柄不定芽诱导率分别为94.17%、65.83%,单位愈伤组织分化芽数分别为3、1.87个;最佳生根培养基为1/2 MS+0.4 mg/L NAA+0.2 mg/L IBA+0.5 g/L碳粉+20 g/L蔗糖+5 g/L琼脂,生根率为97.50%,平均生根数为3.0条,平均根长为3.0 cm;炼苗10 d后,移栽到配比为表土∶椰砖营养土∶黑沙土=1∶2∶1的混合基质中,移栽成活率达到91.7%。