Improving micropropagation of some grape cultivars via boron, calcium and phosphate

BACKGROUND: An efficient regeneration protocol is a priority for the successful application of plant biotechnology. Grape nodal explants were used to develop a micropropagation protocol for Thompson Seedless and Taify cvs. Explants were cultured on MS medium supplemented with Kinetin or benzylaminopurine (BA) and indolebutyric acid (IBA). RESULTS: For both cultivars, axillary buds were grown, only, on a medium enriched with kinetin, moreover, shoot tip necrosis and callus formation were observed on Thompson Seedless cv. cultures grown on a medium with BA. Supplementing the growth medium with 100 mM (boron) B and 2.5 mM (calcium) Ca successfully help overcome these phenomena. The highest regenerated shoot numbers (14 and 6.2 explant 1 ) for Taify and Thompson Seedless cvs., respectively, were on media supplemented with 13.2 mM BA + 4.9 mM IBA and BA 13.2 mM + 5.8 mM IBA, respectively. Moreover, these media supported the developing shoots to have the heaviest dry weights (1.46 and 0.72 mg explant 1 ) for Taify and Thompson Seedless cvs., respectively. Thompson Seedless cv. regenerated shoot numbers and their dry weights were significantly increased by increasing the MS medium PO4 concentration. However, these two parameters were significantly decreased for Taify cv. Developing shoots were elongated and rooted on MS medium enriched with 4.9 mM, IBA 100 mM B and 2.5 mM Ca. Plantlets were acclimatized and successfully transferred to the greenhouse conditions. CONCLUSIONS: A novel promising protocol for Thomson Seedless and Taify cvs. micropropagation using single nodes has been developed.

Critical vitrification steps towards survival of Garcinia hombroniana (Clusiaceae) shoot tips after cryopreservation / Etapas críticas de vitrificación para la supervivencia de ápices caulinares de Garcinia hombroniana (Clusiaceae) después de crioconservación

Abstract A cryopreservation protocol was developed for in vitro shoot tips of Garcinia hombroniana using the vitrification technique. Four critical steps in the technique were investigated, namely preculture, loading, dehydration with Plant Vitrification Solution 2 (PVS2), and unloading. Shoot tips precultured for 48 h gave significantly higher survival (75 %) compared to 24 h preculture (50 %) after cryopreservation. Treatment with 1 M glycerol plus 0.4 M sucrose as a loading solution gave higher survival (45.83 %) compared to the other treatments (0.4 M sucrose + 2 M glycerol; 0.4 M sucrose). Shoot tips dehydrated with PVS2 for 25 min gave the highest survival after immersion in liquid nitrogen. Stepwise PVS2 treatment for 15 min with 50 % PVS2 followed by 10 min with 100 % PVS2 solution improved survival of the shoot tips after cryopreservation (41.67 %). Murashige and Skoog medium with 0.4 M sucrose gave significantly higher survival (66.67 %) than MS with 1.2 M sucrose (25 %) as an unloading solution. Water content was shown to decrease throughout the whole vitrification steps from 6.83 ± 1.66 g g-1 dw for fresh shoot tips down to 2.93 ± 0.28 g g-1 dw after PVS2 treatment. Further study on each step including recovery medium is required to improve the survival. Nevertheless, the present study showed the potential of using the vitrification technique for cryopreservation of G. hombroniana. Rev. Biol. Trop. 66(3): 1314-1323. Epub 2018 September 01.

Resumen Se desarrolló un protocolo de crioconservación in vitro para ápices caulinares de Garcinia hombroniana mediante la técnica de vitrificación, con cuatro etapas críticas: precultivo, carga de crioprotector, deshidratación con solución de vitrificación vegetal 2 (PVS2), y descarga. Ápices precultivados por 48 h sobrevivieron más (75 %) que los de 24 h (50 %) después de la crioconservación. El tratamiento con glicerol 1 M más sacarosa 0.4 M como solución de carga permitió mayor sobrevivencia (45.83 %) que los otros tratamientos (sacarosa 0.4 M + glicerol 2 M; sacarosa 0.4 M). Los ápices deshidratados con PVS2 por 25 min registraron la mayor sobrevivencia tras inmersión en nitrógeno líquido. El tratamiento gradual por 15 min con solución de PVS2 al 50 %, seguido por 10 min al 100 %, mejoró la sobrevivencia de ápices tras la crioconservación (41.67 %). El medio Murashige-Skoog (MS) con sacarosa 0.4 M produjo una sobrevivencia significativamente mayor (66.67 %) que MS con sacarosa 1.2 M (25 %) como solución de descarga. El contenido de agua disminuyó a lo largo del proceso de vitrificación desde 6.83 ± 1.66 g g-1 peso seco, en ápices frescos, hasta 2.93 ± 0.28 g g-1 peso seco después del tratamiento con PVS2. Se requiere de más investigación sobre cada etapa, incluyendo el medio de recuperación, para mejorar la tasa de sobrevivencia. Sin embargo, este estudio muestra el potencial de la vitrificación para la crioconservación de G. hombroniana.

A Review on In vitro Regeneration of Ginger: Tips and Highlights

Ginger (Zingiber officinale) is one of the oriental spices, widely used worldwide for multiple purposes. It is applied as an important ingredient in Ayurvedic preparations from time immemorial. Tissue culture is a practice that is utilized to propagate plants from cells or tissue under sterile conditions. This study is directed to create a review of the successful and reproducible convention for in vitro recovery of ginger with emphasis on effective initial culture establishment. Furthermore, it has dealt with the appropriate explant size and effectiveness of media quality on micropropagation of ginger. The current study recommends that the medium containing Benzyl Amino Purine (BAP) can be used for inducing shoot development of ginger. Among the diverse explants, shoot tips give the quickest response for starting development and the highest number of multiple shoots are produced. As well, it is demonstrated that a survival rate and proper shoot/root expansion can be obtained through the tissue culture methods. Emphasizing the tips and recommendations, this study would be a route-map towards time and cost saving for producing a better quality of ginger.

Influence of Plant Growth Regulators on In Vitro Shoot Multiplication and Plantlet Formation in Cassia angustifolia Vahl

ABSTRACTAn effective and improved plant regeneration system was successfully developed using shoot tip explants taken from a two years old mature plant of Cassia angustifolia. The effect of different cytokinins, [6-benzyladenine (BA), Kinetin (Kin) and thidiazuron (TDZ)] at different concentrations (0.5-10 µM) were evaluated as augmented with Murashige and Skoog (MS 1962) medium. Among all the cytokinins tested, TDZ (5.0 µM) was optimum in inducing multiple shoots as compared to BA and Kin. The rate of shoot multiplication was increased when optimal concentration (5.0 µM) of BA and Kin was tested with different concentration (0.1-1.0 µM) of Indole-3- acetic acid (IAA). Among all the combinations tested, the maximum rate of shoot multiplication was obtained on MS medium supplemented with 5.0 µM BA and 0.5 µM IAA. The number of the shoots and shoot length developed in TDZ was increased when transferred to MS medium devoid of TDZ. After every subculture, rate of the shoot multiplication and shoot length showed increment and continued even after fifth subculture without any decline rate. In vitro rooting in regenerated shoots were best obtained in half-strength MS medium supplemented with 2.0 µM indole-3- butyric acid (IBA). Plantlets with well-developed shoot and roots were successfully hardened off in earthen pots containing garden soil and grown in greenhouse with 80% survival rate.

Direct multiple shoots proliferation of black night shade (Solanum nigrum l.) from shoot tip explants induced by thidiazuron .

The present investigation was undertaken in Black Night Shade (Solanum nigrum L.) which is an important medicinal plant. Direct multiple shoots proliferation was achieved from shoot tip. The shoot tips were cultured on MS medium fortified with Thidiazuron (TDZ) (1.0-7.0 mg/L) for multiple shoot induction. Multiple shoots proliferation was best observed at 3.0 mg/L TDZ from the shoot tip explants within three weeks of culture. Shoot number per explant ranged between 2 and 10. Individual shoots were aseptically excised and sub cultured in the same media for shoot elongation. The elongated shoots were transferred to Indole Acetic Acid/Indole Butyric Acid (IAA/IBA) (0.5mg/L–2.0mg/L) for root induction. Rooting was observed within two weeks of culture. Rooted plantlets were successfully hardened under culture conditions and subsequently established in the field conditions. The recorded survival rate of the plants was 86%. Plants looked healthy with no visually detectable phenotypic variations.

Pattern of Plant Regeneration from Shoot Tip Explants of Pigeonpea (Cajanus Cajan L Milllsp) var LRG-41.

An efficient direct shoot bud differentiation and multiple shoot induction from shoot tip explants of pigeon pea (Cajanus cajan L.) has been achieved. The frequency of shoot bud regeneration was influenced by the type of explants, genotype and concentrations of cytokinin. Explants viz. shoot tip isolated from 10 day old seedlings showed better explants response Explants were cultured on Murashige and skoog (MS) medium augmented with different concentrations of BAP and NAA. Among the various concentrations tested, 2.0mg/l BAP (Benzyl amino purine) and 0.1 mg/l Napthalene acetic acid (NAA) were found to be the best for maximum shoot bud differentiation. Percentage, as well as the number of shoots per explant showing differentiation of shoot buds was higher on MS media supplement with BAP and optimal BAP concentration for shoot regeneration was 2mg/l. The elongated shoots were successfully rooted on MS medium containing different concentrations of auxins. Among them indole buteric acid (IBA) at 1.0mg/l induced maximum frequency of rooting. Regenerated plants were successfully established in soil where 91% of them have been developed into morphologically normal and fertile plants. This method can thus be advantageously applied in the production of transgenic pigeon pea plants.

Efficient Micropropagation of Spilanthes acmella (L.) Murr.: A Threatened Medicinal Herb.

The present study describes an efficient and reproducible protocol for micropropagation of S. acmella. Shoot tips taken from 3 week-old aseptic seedlings were cultured on Murashige and Skoog (MS) semi-solid medium supplemented with different concentrations of TDZ. Among various concentrations, 0.25 μM TDZ was found to be optimum for shoot regeneration as it induced a maximum of 30.0 shoots per explant however with retarded growth (1.0 cm). Among different volumes of culture media, 15 ml liquid culture medium favored best response wherein a maximum of 80.2 shoots per explant with an average shoot length of 7.0 cm were induced after 6 week of subculturing. Successful in vitro rooting was induced on 2.5 μM NAA containing half-strength MS medium. Almost 96% rooted plants successfully transferred and acclimatized ex vitro under green house conditions. Morphological and physiological parameters compared with the in vivo-grown seedlings of the same age appeared to be ‘normal’ in respect to the fundamental characteristics examined.

Factors Affecting on the Survival Rate for Shoot-tip Micro-grafting of Citrus medica L.var.sarcodactylis Swingle / 广州中医药大学学报

Objective To lay a foundation for the cultivation of disease-free plantlet of Citrus medica L.var.sarcodactylis Swingle(CMS) by discussion of the factors affecting survival rate for micro-grafted shoot-tip of CMS.Methods With citrus limon seedlings cultured in the dark as the rootstock,shoot-tip of CMS was grafted to the inverse T-shaped cut in the cotyledon of rootstocks.Then an investigation was carried out on the factors which affected the survival rate for micrografted shoot-tip of CMS.The factors included age of rootstocks,scion size,culture medium with different concentrations of phytohormones,and rootstocks with or without cotyledon.Results The survival rate for micro-grafted shoot-tip of CMS was closely related to the factors mentioned above.A higher survival rate was obtained when CMS shoot-tip with 4 pieces of leaf primordia was grafted to the inverse "T" shaped cut in the cotyledon of parental stock(citrus limon seedlings) which was cultured in the dark for 14 days,and when the citrus limon seedlings were cultivated in MT-based medium with ?-naphthylacetic acid 0.1 mg?L-1 added.Conclusion The optimized micro-grafting conditions for CMS shoot-tips improve the propagation of micro-grafted plantlet and the survival rate of micro-grafted shoot-tip of CMS.

Vitrified cryopreservation and regeneration of shoot-tips for Citrus medica var.sarcodactylis / 中草药

Objective To provide a new approach of preservation for germplasm resources of Citrus medica var.sarcodactylis(CMS).Methods A micrografting test was made on CMS shoot-tips after vitrified cryopreservation,resulting in living shoot-tip.Results First the CMS shoot-tip is inoculated in medium consisting of MS,5%DMSO,and 5%sucrose for 48 h preincubation.Then the shoot-tips were cut off 3—4 mm long and fore-treated by 60%PVS_2 at room temperature(25℃) for 30 min.After that,they were treated by PVS_2 at 0℃for another 80 min and conserved in liquid nitrogen for 24 h.Next the shoot-tips were defrosted by aqueous bath at 40℃and cleaned twice at room temperature(25℃) by MT minimal medium with additive 1.2 mol/L sucrose,10 min once.Finally data collected recorded a higher survival rate of 81.25%by TTC inspectation and a better regeneration rate of 52.94%if the CMS shoot-tips had been grafted on darkly-cultured parental stock,growing,and differentiating normally afterwards.The plantlets detected by polymerase chain reaction(PCR) showed that the pathogens of Huanglong disease had been removed.Conclusion Therefore a conclusion is made that the approach of vitrified cryopreservation may be applied to the preservation of CMS germplasm resources.

Shoot tip culture and virus detection of Dioscorea bulbifera / 中草药

Objective Effects of different factors on shoot tip culture in vitro were studied by using Dioscorea bulbifera as test material to find the media suitable to the shoot tip differentiation of D.bulbifera and the method of virus delection.Methods Plant tissue culture method was used in shoot tip culture and the symptom observation method,instruction plant method,and RT-PCR method were used in virus detection of virus-free plantlets.Results The best disinfection method for D.bulbifera shoot tips was firstly disinfected for 30 s with 70% alcohol and then disinfected for 12 min with 0.1% HgCl2;It is better for D.bulbifera to cut shoot tips in 0.5 mm length after 37 ℃ heat treatment for 7 d;The best proliferation medium of D.bulbifera shoot tips was MS+KT 2 mg/L+NAA 0.5 mg/L;The best rooting medium of regeneration buds from D.bulbifera shoot tips was 1/2 MS+NAA 0.5 mg/L;The best matrix of regeneration plantlets from D.bulbifera shoot tips was perlite-vermiculite (2∶1);RT-PCR Method was primary mean to detect potato virus Y (PVY) of regeneration plantlets from D.bulbifera shoot tips,and average rate of PVY-free was 86.5% by RT-PCR.Conclusion The virus-free culture system of D.bulbifera shoot tips is established for the first time,providing a technological basis for the rapid propagation and factory production of D.bulbifera virus-free plantlets.