Percentage Of Shoot Induction Research Articles

PERBANYAKAN TUNAS Gyrinops versteegii (Gilg.) Domke

Gyrinops versteegii (Gilg.) Domke) is a member of Thymellaceae having an important role as agarwood source that is potential to develop. Agarwood is a non timber forest product (NTFP) with a high economic value. Gyrinops versteegii (Gilg) Domke was an endemic and rare plants, however, it is cultivated on the island of Lombok. The study of shoot multiplication from single node stem and cotyledon node explants to shoot produced was conducted to find out the best method to shoot multiplication. Modification of Murashige and Skoog (MS), Gamborg (B5) dan Woody Plant Medium (WPM) supplemented with 8.0 g/l agar, 30 g/l sucrose and vitamin (0.1 mg/l Thyamine, 0.5 mg/l Nicotinic acid, 0.5 mg/l Pyridoxine dan 2 mg/l Glysin) and 10 g/l myoinositol were used as growth medium treatments. The experiment was conducted on two stages i.e. germination and shoot multiplication. Shoot induction was conducted on modification of MS medium supplemented with Benzyl Adenin (BA) 0.5; 1.0 and1.5 mg/l. The multiplication of shoot conducted on modification of MS medium, WPM and B5 medium supplemented with 0.5; 0.75 dan 1.0 mg/l Benzyl Adenin (BA). For every treatment there were ten samples (bottles) with one explants in each bottle. The observation was conducted on shoot induction percentage, number of shoot and visual performance of culture. The result showed that modified MS + BA 0.5 mg/l was the best for shoot induction from single node stem explants with the average of 1.6 shoots on 12 weeks. The treatment of modified MS + BA 0.75 mg/l was the best for shoot multiplication from single node stem explants with the average of 5.7 shoots on 12 weeks. The treatment of modified WPM + 0.5 mg/l BA was the best for shoot multiplication from cotyledonery node explants with the average of 4.6 shoots on 12 weeks. The visual performance of shoot resulted was normal.

Micropropagation of Erodium sibthorpianum subsp. sibthorpianum, an endemic threatened species of Uludağ Mountain (Bursa-Turkey)

Uludağ Mountain is one of the Important Plant Areas of Turkey. Its native plant species are threatened by various human and environmental factors. In this study, 2 different micropropagation techniques, with Murashige Skoog (MS) and modified MS (ModMS) media, were used for Erodium sibthorpianum Boiss. subsp. sibthorpianum. Mineral composition was developed according to the plant requirements. Shoot tips were cultured on these media. Seedlings, obtained from in vitro germinated seeds, were used as explant sources in micropropagation studies. The highest percentage of shoot induction (11.33 shoots/explant) was determined in 1 ppm 6-benzyl amino purine and plant growth regulator-free MS medium (35.97 mm shoot length). Elongated shoots were successfully rooted in the ModMS medium with different naphthalene acetic acid concentrations. Shoots with regenerated roots were transferred to the greenhouse. This protocol could be used for conservation studies of endemic and endangered plant species.

Silicon Promotes Adventitious Shoot Regeneration and Enhances Salinity Tolerance ofAjuga multifloraBunge by Altering Activity of Antioxidant Enzyme

We investigated the effect of Si concentration on shoot regeneration and salinity tolerance of Ajuga multiflora. Addition of Si to the shoot induction medium significantly increased the frequency of shoot induction. The average number of shoots regenerated per explant decreased on the medium containing NaCl alone, while there was less decrease when the shoot induction medium was supplemented with both NaCl and Si. The shoot induction percentage increased linearly with increasing concentration of Si in the NaCl containing medium. Addition of Si to the shoot induction medium significantly increased SOD, POD, APX, and CAT activity in regenerated shoot buds as compared with the control. The inclusion of Si to the NaCl containing medium significantly increased the SOD activity in leaves and roots, while it decreased POD, APX, and CAT activity in both organs. Scanning electron microscopic analysis showed that there are no distinct differences in the structure of stomata between the control and Si-treated plants. However, NaCl treatment significantly affected the structure and number of stomata as compared to the control. Wavelength dispersive X-ray analysis confirmed the high Si deposition in trichomes of plants grown in the Si containing medium but not in plants grown in the medium without Si.

Genetic transformation of hop (Humulus lupulus L. cv. Tettnanger) by particle bombardment and plant regeneration using a temporary immersion system

The efficiency of micropropagation of double-node shoots of hop (Humulus lupulus L. cv. Tettnanger) was evaluated using semi-solid and liquid culture medium in RITA® temporary immersion bioreactors. The highest fresh and dry weight of shoots, average number of shoots, and multiplication rate were obtained using the RITA® system, whereas the longest shoots were obtained on semi-solid medium. Moreover, shoot length was affected significantly by the inoculum density of double-node shoots in RITA® vessels. In addition, the RITA® bioreactors were suitable for shoot induction from organogenic calli. The percentage of shoot induction and the shoot fresh and dry weights were significantly higher in the RITA® system than in semi-solid medium. The age of organogenic calli and inoculum density significantly affected the induction of shoots from organogenic calli. The optimum conditions for DNA delivery into hop organogenic calli using the biolistic particle delivery system were also determined. Organogenic calli were bombarded with the plasmid pSR5-2 (gusA and nptII) varying helium pressure (900, 1,100, or 1,350 psi) and target distance (6, 9, or 12 cm). The highest gusA transient activity was obtained using a pressure of 900 psi and a target distance of 6 cm. For stable genetic transformation, 3-wk-old organogenic calli were bombarded with the plasmid pCAMBIA1303 (gusA, mgfp5, and hpt) using these optimum conditions. Stable gusA expression was observed in organogenic calli and shoots after 4 wk of culture on selection medium containing 2.5 mg l−1 hygromycin. The presence of the mgfp5 gene in the hop genome was confirmed by PCR.

Direct Shoot Organogenesis in Murraya paniculata (L.) Jack: A Prerequisite for Genetic Transformation

An efficient in vitro regeneration system through direct shoot organogenesis was established for Murraya paniculata (L.) Jack (Orange Jessamine). Epicotyls, leaves, roots, and cotyledons from in vitro-germinated seedlings and several plant growth regulators (PGRs) were evaluated for their effects on plant regeneration. Longitudinally cut epicotyl segments were observed to be the optimal explants followed by uncut epicotyls (not longitudinally cut). Roots, leaves, and cotyledons were not suitable as explants as a result of little or no shoot induction. Adventitious shoot induction was enhanced by the addition of 6-benzyladenine (BA). The highest percentage of shoot induction (87%) and the greatest number of shoots per explant (12.7) occurred on Murashige and Skoog (MS) medium supplemented with 15 μM BA from longitudinally cut epicotyls followed by 5.2 shoots per explant from uncut epicotyls. Optimal concentration of gibberellic acid (GA3) for shoot elongation was observed to be 15 μM. Eighty-five percent of the regenerated shoots produced roots with an average of three roots per shoot on MS medium supplemented with 5 μM indole-3-butyric acid (IBA). Our protocol for direct shoot organogenesis can potentially lead to the development of a robust method for production of transgenic plants of M. paniculata through Agrobacterium-mediated genetic transformation.

In vitro propagation of Etlingera elatior (Jack) (torch ginger)

An efficient protocol for complete plant regeneration from suckers of Etlingera elatior (Jack) has been developed. The addition of N6-benzyl amino-purine (BAP) (0, 13.32, 22.20, 31.08, and 44.40μM) to the culture medium comprising Murashige and Skoog (MS) basal salts, 3% sucrose, 0.4% Gelrite™ did not show any significant effects on percentage of shoot induction and mean number of shoots produced. Various concentrations of BAP (0, 13.32, 22.20, 31.08, and 44.40μM), 6-furfurylaminopurine (kinetin) (0, 13.95, 23.25, 32.55, and 46.50μM) and N6-(2-isopentenyl) adenine (2-iP) (0, 14.76, 24.6, 34.44, and 49.2μM) were tested for shoot multiplication. BAP at all levels were found suitable for the multiplication of shoot. However, the low level of 13.32μM BAP was chosen as the best concentration of BAP due to economic feasibility. The best root proliferation was observed on MS medium without plant growth regulator (PGR). Assessment of various potting media for acclimatization showed medium containing soil:sand:peat moss (1:1:1) produced high survival of plantlets, number of leaves produced per plant and the plant height. This protocol provides a basis for future studies on mutation induction, genetic engineering, and could be applied for large-scale multiplication of this valuable plant.

Propagation of goldenrod (Solidago canadensis L.) from leaf and nodal explants

Goldenrod (<em>Solidago canadensis </em>L.) is an invasive plant species in many countries except North America but a cut-flower species worldwide. There is a need to generate and propagate goldenrod clones efficiently for research and commercial purposes. A callus induction and plantlet regeneration system was developed by studying the influence of explant type and different concentrations of plant growth regulators. The highest callus production from leaf segments was obtained on Murashige and Skoog’s medium (MS medium) supplemented with 1.0 mg/L naphthalene acetic acid (NAA) and 1.0 mg/L 6-benzylaminopurine (BA). Adventitious shoots could be regenerated directly from leaf explants without an intermediate callus phase with the highest shoot induction percentage of 87.2%. The largest number of adventitious shoots per leaf explant (3.2) was obtained on MS medium supplemented with 0.4 mg/L NAA and 2.0 mg/L BA. MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L BA was the best medium for axillary shoot regeneration from nodal segments. The highest root number and longest roots occurred on half-strength MS without the addition of any growth regulator. Rooted plantlets were then transferred to a soil-based growth medium, placed in a greenhouse, and acclimatized with 100% success. All surviving plants grew normally without showing any morphological varia­tion when compared to those grow from seed. This regeneration protocol may be used to produce certain biotypes of goldenrod suitable for genetic transformation rapid propagation of goldenrod for commercial purposes or for screening fungi and toxins as potential biocontrol agents against this weed.

In vitro adventitious shoot regeneration and acclimatisation of Brassica oleracea subsp. italica cv. Green Marvel

Cotyledonary explants of Brassica oleracea subsp. italica (broccoli) cv. Green Marvel were cultured on Murashige and Skoog (MS) medium containing different combinations of the growth regulators 6- benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) for shoot regeneration. The optimal medium for inducing shoots contained 3 mgl -1 BAP and 1 mgl -1 NAA, which produced a shoot induction percentage of 53.33% and a mean number of 0.43 shoot per explant. The shoots were subsequently rooted in MS medium that contained 0.2 mgl -1 of indol-3-butyric acid (IBA). Different potting media were assessed during plantlet acclimatization. The highest percentage of plant survival (83.33%) was on the medium that contained sand and soil (1:1), while maximum root length (4.37 cm) and plant height (7.87 cm) were attained in potting medium that consisted peat moss, perlite and vermiculite (3:1:1). Key words : Brassica oleracea, broccoli, 6-benzylaminopurine, α-naphthalene acetic acid, indole-3- butyric acid.

Micropropagation of Plumbago zeylanica L.

vitro propagation of Plumbago zeylanica was investigated to develop reliable protocols for direct and indirect shoot regeneration. Axillary shoot multiplication, callus induction and shoot regeneration from callus culture was obtained on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. A maximum number of shoots (38 ± 1.3 per explant) was obtained from nodal explants when cultured on MS liquid medium supplemented with 1.0 mg·L -1 BAP, 0.5 mg·L -1 IBA and 2.0 mg·L -1 adenine sulfate. The highest percentage of callus induction was obtained when stem explants cultured on MS medium supplemented with 2.0 mg·L -1 BAP and 1.5 mg·L -1 IAA. The greatest percentage of shoot induction (100%) with a mean of 34.2 shoots obtained from callus was cultured on MS medium supplemented with 0.75 mg·L -1 BAP, 1.0 mg·L -1 IAA, NAA and adenine sulfate each. Regenerated shoots were rooted best on half-strength MS medium containing 0.5 mg·L -1 NAA and 3% (w/v) sucrose. The regenerated plantlets were acclimatized in the culture room and successfully transferred in soil.

Influence of plant growth regulators on axillary shoot multiplication and iron source on growth of Scrophularia takesimensis Nakai - a rare endemic medicinal plant

An efficient protocol for the in vitro propagation of Scrophularia takesimensis, a rare endemic medicinal plant, is described. Shoot multiplication was induced by culturing nodal explants on MS medium containing 3% (w/v) sucrose, 0.8% (w/v) agar, and different concentrations and combinations of plant growth regulators. The greatest percentage of shoot induction was achieved when nodal explants were cultured on MS medium supplemented with 2.0 mg l-1 BAP and 1.0 mg l-1 IAA with an average of 16 shoots per explant. The microshoots were separated from the multiple shoots and sub-cultured onto MS medium with 3% (w/v) sucrose and 0.8% (w/v) agar for further growth, and rooting. The plantlets growth was slow and often showed chlorosis on leaves. This problem was overcome by transferring microshoots to MS medium modified by increasing FeSO4 (55.6 mg
L-1) and Na2EDTA (74.52 mg
L-1) salts concentration. The iron concentration had a significant effect on chlorophyll content of the leaves. Chlorophyll content was increased by increasing FeSO4 and Na2EDTA salts concentration. Maximum rooting was obtained on modified MS medium supplemented with 1.0 mg l-1 IBA. The in vitro-grown plantlets were successfully established in the field with 96% of survival. This protocol could be utilized for conservation and clonal propagation of this economically important plant.

Propagación in vitro de Psidium guajaba mediante organogénesis directa a partir de segmentos nodales

Se indujeron múltiples brotes mediante organogénesis directa a partir de segmentos nodales de 10 genotipos diferentes de guayaba. Para ello se estableció un sistema de propagación clonal in vitro combinado con inducción rápida de brotes ex vitro para propagar árboles élite. La utilización de segmentos nodales permitió obtener en poco tiempo brotes adventicios adecuados para multiplicación masiva. La respuesta in vitro de los genotipos fue evaluada usando los medios de cultivo MS (Murashige y Skoog, 1962), Mc (Mascarenhas) y WPM (Woody Plant Medium) suplementados con 0,1 mg•L-1 de ácido indolácetico (AIA) y 0,25 mg•L-1 de bencilaminopurina (BAP). El procedimiento de desinfección con hipoclorito de sodio previno eficientemente la contaminación de los explantes después de la inoculación en el medio de cultivo. El mayor porcentaje en la inducción de brotes se logró con 0,25 mg•L-1 de BAP. Los segmentos nodales presentaron de 1 a 2 brotes adventicios por explante después de 15 días de inoculados y de 3 a 7 brotes a los 30 días después del inicio del cultivo. Una vez individualizados los brotes se usaron en una nueva fase de multiplicación masiva en la que se probaron cuatro sustratos diferentes durante el enraizamiento y el endurecimiento. Esta metodología permitió la propagación in vitro de guayaba cuatro semanas después del inicio del cultivo. Los mejores resultados se lograron con el medio WPM que permitió obtener las primeras plántulas enraizadas dos semanas después de la transferencia al sustrato de enraizamiento.

High-frequency embryogenesis, regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

Abstract High-frequency somatic embryogenesis and shoot regeneration of broccoli ( Brassica oleracea var. italica ) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO 3 on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l −1 , 6-BA at 3.0 mg l −1 , AgNO 3 at 4.0 mg l −1 and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l −1 , respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.

Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterlessgus::nptII fusion gene based vector

We have generated putative promoter tagged transgenic lines in Arachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated by Agrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4mg/l in combination with 0.1 mg/l alpha -napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age, Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated with Agrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in beta-glucuronidase (GUS) assays. Among the in vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T 0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T 0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T 0 and corresponding T 1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.

Evaluation of Runner and Virginia Market Types for Tissue Culture Responses

Abstract Studies on somatic embryogenesis and organogenesis of peanut (Arachis hypogaea L.) derived from mature zygotic embryo-derived leaflets of 10 cultivars and two breeding lines representing both runner and Virginia market types were conducted using tissue culture procedures originally optimized for the Spanish market type, J.L. 24. The somatic embryogenesis protocol used was a multi-step process which included embryogenic mass induction, embryo development, germination, and conversion. The percentage of embryogenic mass induction, embryo development, and conversion was genotype-dependent. However, the number of embryos produced per explant and germination to plantlets was genotype-independent. SunOleic® 95R consistently ranked highest for its response during all stages of embryo development, and runner market types were generally more responsive than Virginia market types. The percentage of shoot induction and the number of shoot buds per explant during organogenesis were genotype-dependent, and runner market types were more responsive for organogenesis than Virginia market types. Genotypes with a high embryogenic response also were highly responsive for organogenesis.

Applicability of the co-inoculation technique using Agrobacterium tumefaciens shooty-tumour strain 82.139 in silver birch

In the co-inoculation technique, genetic transformation is performed using a mixture of Agrobacterium strains – shoot regeneration is induced by the wild-type strain 82.139, while the transferable genes are provided in a binary plasmid by another, non-oncogenic Agrobacterium strain. The aim of the present work was to study the applicability of co-inoculation under both in vitro and greenhouse conditions for in planta transformation in silver birch (Betula pendula Roth). In addition to the original method, several modifications of the technique including an genetically engineered 82.139 strain harbouring the binary pGUSINT were tested. The co-inoculations resulted in a gall formation frequency of 52–94% with greenhouse seedlings, and 4–63% with tissue-cultured plantlets, the shoot induction percentage varying by 0–13 in the greenhouse and 42–75 in vitro. PCR analysis verified that the majority of the regenerated material was non-transgenic, with a few individuals showing integration of the oncogenic T-DNA. According to the histochemical tests, however, some of the numerous differentiating buds and small shoots on gall tissues were transgenic, and contained the GUS reporter gene. The results show that it would have been necessary to apply selection pressure during differentiation in order to recover shoots transformed with the desired genes from the binary plasmid. The morphology and growth of all the regenerated plantlets was normal, suggesting that the oncogenic T-DNA was not expressed even though it was present. In conclusion, it was possible to obtain transgenic silver birch plantlets using the A. tumefaciens strain 82.139, but the co-inoculation method is not directly applicable as in planta transformation protocol.

Effect of various growth regulators on shoot regeneration of sugarcane

Sugarcane (Saccharum spp. hybrid cv. CP 84-1198) embryogenic calluses were induced from young leaves cultured on modified Murashige and Skoog basal medium supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid. Five concentrations, 0.5, 1.0, 2.5, 5.0, and 10.0 μM, of five different growth regulators, 6-benzylaminopurine, kinetin, 6-γ,γ-(dimethylallylamino)purine, zeatin, and thidiazuron, were tested with or without 22.5 μM α-naphthaleneacetic acid to compare their ability to induce regeneration from embryogenic callus. After 4 wk on medium, the percentage of shoot meristem induction was evaluated, and after 10 wk the total number of shoots produced, as well as the percentage of shoots greater than 1 cm in length, was obtained. Although it had the lowest percentage of elongated shoots, medium containing thidiazuron alone performed better than all other growth regulators tested, with the highest percentage of shoot induction and the largest number of shoots, particularly at a concentration of 2.5 μM.