Number Of Multiple Shoots Research Articles

Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development

An in vitro plant regeneration protocol was successfully established for Cymbidium bicolor an epiphytic orchid by culturing seeds from green pods. Immature seeds were germinated on four basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC) orchid medium, Knudson C modified Morel (KCM) medium and Lindemann orchid (LO) medium. Seed germination and protocorm development was significantly higher in LO medium (96.6 %) followed by KCM (89 %), MS (77.5 %) and KC (62.7 %) media after 56 days. For multiple shoot induction the protocorms were transferred to B5 medium supplemented with cytokinin. Among the various cytokinins tested, BAP (4.42 μM) induced maximum (27.59) number of multiple shoots per explant. IBA was effective in inducing healthy roots. Tissue-cultured protocorms and seedlings of C. bicolor were inoculated with AC-01 fungal strain (Moniliopsis sp.) isolated from the mycorrizal roots of an epiphytic orchid Aerides crispum. Mycorrhizal fungi significantly enhanced number of roots, root length and shoot number.

Study of the effect of physical state of medium and different concentrations of sucrose, ferric ethylenediamine- tetraacetic acid (FeEDTA) and CuSO4 in enhancing the micropropagation system of Stevia rebaudiana

Stevia rebaudiana is a sweet herb native to Paraguay and Brazil. Its leaves are well known for containing sweet glycosides which are intensely sweet compounds with zero caloric value. A comparison of effect of state of medium on the in vitro growth of S. rebaudiana was studied under controlled conditions. Different concentrations of benzyl amino purine (BAP) and kinetin (Kin) (1, 2 and 3 mg/L) were used for the purpose. Explants inoculated in liquid media placed on orbital |shaker showed the best results for the vigorous growth of the plant in all the hormone treatments. In all the hormone treatments, shaking media gave the maximum values of number of multiple shoots as well as shoot length. Shaking cultures also showed response in minimum days of inoculation as compared to solid and static liquid cultures of honey leaf, S. rebaudiana. Medium conditions were also optimized for the maximum micropropagation potential of the S. rebaudiana explants in shaking medium by adding different concentrations of Fe- EDTA, CuSO4 and sucrose. Optimal concentrations were determined as 60 g/L sucrose, 9 ml/L Fe-EDTA and 27.5 µg/L CuSO4. Key words: Benzyl amino purine (BAP), kin, liquid medium, Stevia rebaudiana, CuSO4, ferric ethylenediamine- tetraacetic acid (FeEDTA), sucrose.

In vitro propagation of Cuphea procumbens Orteg. and Evaluation of genetic fidelity in plantlets using RAPD marker

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cuphea procumbens Orteg. using cotyledonary node explants excised from 15 days old aseptic seedlings. A range of cytokinins were investigated for multiple shoot regeneration. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at a concentration of 2.5 μM was effective in inducing multiple shoots. The highest number of multiple shoots (9.33 ± 0.60) and maximum average shoot length (4.16 ± 0.44 cm) was standardized on MS medium supplemented with 2.5 μM BA alongwith 0.5 μM NAA. Addition of 200 mg/l Casein hydrolysate (CH) to the shoot induction medium enhanced the growth of regenerants. Rooting of in vitro regenerated shoots was best achieved on 1/2 strength MS medium. The in vitro raised plantlets with well developed shoots and roots were hardened, successfully established in earthen pots containing garden soil and maintained in greenhouse with 80% survival rate. Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate the genetic stability among in vitro regenerated progenies. All RAPD profiles from the micropropagated plants were monomorphic and similar to control plant. These results suggests that the culture conditions used for the axillary bud proliferation are appropriate for clonal propagation of this medicinally important plant as they do not appear to interfere with genetic integrity of in vitro regenerated plants. The described method can be successfully employed for large-scale multiplication and in vitro conservation of C. procumbens.

STUDIES ON PRE ACCLIMATIZATION TREATMENTS TO IMPROVE POST ACCLIMATIZATION SURVIVAL IN STEVIA (STEVIA REBAUDIANA BERT.)

The special conditions during in vitro culture result in the formation of plantlets of abnormal morphology, anatomy and physiology. After ex vitro transfer, these plantlets might easily be impaired by sudden changes in environmental conditions, and so need a period of acclimatization to correct the abnormalities. The large number of plants lost or damaged during acclimatization often restricts application of micropropagation for rapid multiplication of plants. Acclimatization continues to be a major bottleneck in the micropropagation of stevia too with lower success reported. In the present investigation, pre acclimatization treatments such as dipping the explants in high concentration cytokinin (benzyl adenine, BA) before culture and use of growth retardants that inhibit gibberellin biosynthesis (ancymidol) in culture were used to study their effect on post acclimatization survival of micropropagated plantlets. Dipping of explants in high concentration BA resulted in an increase of number of multiple shoots and number of leaves per shoot and survival percentage after acclimatization. But a significant reduction was noted in internode length and shoot height. Dipping the explants with 100 mg/L BA before culture resulted in highest survival after acclimatization. Ancymidol reduced the length of the shoot, number of leaves, and internodal length considerably. Survival percentage increased with concentration until 3 mg/L ancymidol and reduced with further increases in ancymidol concentration. This reduction might be because the excessive reduction in growth characteristics, which made the plants too fragile to withstand the pressure of acclimatization. The use of 3 mg/L resulted in a higher number of multiple shoots and average growth parameters and a better survival percentage (75.80% - increase of 25.29% over the control - 60.50%).

MULTIPLE SHOOT REGENERATION FROM THE CULTURES OF ADHATODA VASICA NEES.

The alkaloids present in Adhatoda vasica Nees. namely vasicine and vasicinone are responsible for its pharmacological property. Due to exploitation of the plant for various medicinal uses, there is a need to conserve this plant. High frequency and maximum number of multiple shoots were obtained from shoot tip explants harvested during the month of May on MS medium supplemented with BAP (22.20 μm). Rest of the year the explants were turning brown or black after a day of inoculation because of phenolic substances present in the explant. High frequency of rooting was recorded on MS basal medium. The rooted plantlets were hardened and established at 50-60%.

An efficient method for clonal propagation and in vitro establishment of softwood shoots from epicormic buds of teak (Tectona grandis L.)

Softwood shoots were produced from 40 cm long stem segments placed horizontally in flat trays containing sterilized sand under natural light or shade conditions for subsequent rooting and micropropagation studies in teak (Tectona grandis L.). Higher number of shoots (6.17) per log was produced under natural light as compared to shade conditions. Forcing was also better in natural light as compared to shade in terms of shoot length, number of nodes or leaves. For rooting, 2–4 cm long softwood shoots were excised and treated with either indole-3-butyric acid (IBA) or α-naphthyl acetic acid (NAA) at 0, 1000, 2000 or 3000 μmol·L−1 each or with combinations (1000 + 1000, 2000 + 2000 or 3000 + 3000 μmol·L−1) and then placed in flat trays containing autoclaved sand at 25 ± 2°C in 16 h photoperiod at 35 μmol·m−2·s−1. After 28 days, softwood cuttings treated with IBA + NAA (3000 + 3000 μmol·L−1) had highest rooting percentage (89.3%) with 5.5 mean roots. Shoot apex and nodal explants of softwood cuttings were pretreated with 0.1% (w/v) ascorbic acid, boric acid, activated charcoal, citric acid, glutamine or polyvinylpolypyrollidone (PVP) for 24 h to remove phenolic compounds before surface disinfestation. Glutamine (Gl) and PVP were equally effective resulting in 60% establishment of shoot apices on MS medium supplemented with 10 μmol·L−1 6-benzylaminopurine (BAP) + 5 μmol·L−1 NAA. Using shoot apices, highest (42.80) number of multiple shoots with 54.33 mm shoot length were obtained on MS + BAP (8.8 μmol·L−1) + IBA (2 μmol·L−1) after 45 days. Shoots were successfully rooted and acclimatized to greenhouse conditions.

In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants

Pterocarpus marsupium (Bijasal) is a valuable multipurpose forest tree. The regeneration rate in natural habitat is low and the tree is overexploited. An in vitro propagation protocol has been developed from nodal explants obtained from in vitro raised 18-day-old axenic seedlings. The highest shoot regeneration frequency (85%), maximum number of multiple shoots (8.6) as well as length (4.8 cm) were induced from nodal explants on Murashige and Skoog (MS) medium amended with 4.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 20 μM adenine sulphate (AdS). The percentage of shoot multiplication as well as the number of shoots per node remained the same during the first two subculture, afterwards a decline was recorded. Rooting was best induced in microshoots excised from proliferated shoot cultures on semisolid hormone-free half-strength MS medium, after a pulse (dip) treatment for 7 days in half-strength MS liquid medium containing 100 μM indole-3-butyric acid (IBA) and 15.84 μM phloroglucinol (PG). The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 4 weeks before their transfer to a greenhouse, where the established plants showed 75% survival.

Micropropagation and Comparative Study of Chemical Components of Essential Oils of in-vitro and in-vivo Grown Mentha spicata L.

Axenic shoot culture of Mentha spicata L. was established from young shoots of the plant naturally growing in the field. Large number of multiple shoots were observed on MS media supplemented with NAA (0.5ppm) and BAP (1ppm) within eight weeks of culture. Extensive root formation was observed on MS medium supplemented with 1 ppm NAA. Essential oils from both in vitro and in vivo samples showed l- carvone as a major constituent, however, the in vitro samples showed higher concentration of menthol than in vivo samples. <i>Nepal Journal of Science and Technology</i> Vol. 7, 2006

Effect of Urea-Type Cytokinins On The Adventitious Shoots Regeneration From Cotyledonary Node Explant in Thecommon Ice Plant, Mesembryanthemum Crystallinum

Mesembryanthemum Crystallinum (Common Ice Plant) Was Used As A Model Plant To Study The Regulatory Properties of Crassulacean Acid Metabolism (Cam) and Tolerance To Abiotic Stresses. Although Transformation Is A Useful Genetic Approach, It Has Not Been Established in This Species Due To Recalcitrancy For Regeneration. To Establish An Efficient Procedure For Regeneration of M. Crystallinum, We Examined The Effects of Urea-Type Cytokinins, Thidiazuron (Tdz) and Forchlorofenuron (Cppu) On The Adventitious Shoot induction. Adventitious Shoots Were Generated Only From Explants Obtained From The Cotyledonary Node, Not From Explants Obtained From The Cotyledon, Hypocotyl and Roots. Urea-Type Cytokinins, Tdz and Cppu Were More Effective For The induction and The Morphogenesis of Adventitious Shoots Than Adenine-Type Cytokinin, 6-Benzyladenopurine (Ba). We Have Found That The 2.5 Mg L-1 Tdz induced The Largest Number of Multiple Shoots and The Highest Frequency of Adventitious Shoot induction From Single Explant. in Addition, Fewer Hyperhydric Shoots Were Produced On The Medium Containing Tdz Than in That Containing Ba and Cppu in The Presence of 1.0 Mg L-1 Naa. The Regenerated Shoots Rooted On The Ms Medium Within One Month, and The Rooting Was Promoted By Replacing The Agar Medium With Vermiculite Or Florialite. The Fertile Plant With Normal Morphological Properties Was Harvested For Four Months After Sowing. Using The Improved Regeneration Procedure With Tdz, We Successfully introduced A Kanamycin-Resistant Gene (Nptii-Hph) into The Cotyledonary Node Mediated By Agrobacterium Tumefaciens. These Results indicated That This Regeneration Procedure Using Cotyledonary Node Explants and Tdz Could Be Useful For The Genetic Engineering of M. Crystallinum.

An efficient micropropagation system for Celastrus paniculatus Willd.: a vulnerable medicinal plant

A micropropagation protocol was developed for Celastrus paniculatus, a vulnerable medicinal plant. Cultures were initiated from nodal explants collected from young shoots of a 12-year-old plant in MS basal medium. An average of five shoots were produced in MS medium supplemented with 1.5 mg l−1 benzyl adenine (BA) and 0.1 mg l−1 naphthalene acetic acid (NAA) after two subculture cycles with a 30-day interval. Continuous subculture in the same medium for three more cycles resulted in reduction of the number of multiple shoots (2 or 3 shoots), vitrification of the shoots, and callus formation. Vitrification of cultures could be overcome by the use of MS medium supplemented with lower concentrations of BA (0.05mg l−1) and NAA (0.01mg l−1). Among the various rooting trials, ex vitro rooting of shoots with simultaneous hardening was most efficient. The method standardized in the present study is simple, as it eliminated separate steps for in vitro rooting and hardening. Qualitative chemical similarity of the tissue culture regenerants with the mother plant was confirmed using high performance thin-layer chromatographic (HPTLC) profiling.

Reversion of inflorescence development in Euphorbia millii and its application to large-scale micropropagation in an air-lift bioreactor

SummaryAn efficient, simple micropropagation protocol was developed for Euphorbia millii Des Moulines using inflorescence explants and an automated, low cost, air-lift bioreactor system. Inflorescence meristems of the main axis usually reverted to vegetative meristems (a meristematic dome with leaf primordia) 1 week after inoculation on MS medium supplemented with benzyladenine (BA) and indole-3-butyric acid (IBA). Of the various plant growth regulator combinations tested, MS medium with 1 mg l–1 BA and 0.3 mg l–1 IBA proved to be most effective. On this medium the percentage reversion was maximal, and the number of explants producing multiple shoots was the highest. The highest percentage of inflorescence reversion and the highest number of multiple shoots induced per explant were observed when inflorescences from the first stage of development were used. The lowest response was observed on third stage inflorescences. Vegetative shoots obtained from reverted explants of inflorescences were used as inocula for multiplication studies. Comparisons between solid and bioreactor cultures (i.e., continuous immersion with or without a net, or temporary immersion in liquid media using ebb and flood) revealed that shoot multiplication and growth were more efficient using a continuous immersion bioreactor (with a net). In vitro regenerated shoots were cultured hydroponically for 6 weeks; 100% of plants rooted and were acclimatised successfully in growing media containing peat moss:vermiculite:perlite (1:1:1). RAPD DNA marker analysis of micropropagated plants and their mother plant confirmed the genetic uniformity of the regenerants.

Factors influencing shoot regeneration from cotyledons of tetraploid Isatis indigotica fort

An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS) medium was found to be best for promoting shoot regeneration, followed by Gamborg's B5 and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented with 2.0 mgl−1 (8.9 μM) 6-benzyladenine and 1.0 mgl−1 (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl−1 (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage.

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl−1 (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl−1 (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.

Optimization of Media Constituents for Shoot Regeneration from Leaf Callus Cultures of Decalepis hamiltonii Wight. & Arn.

Response surface methodology was utilized in statistical optimization of three quality factors (the number of multiple shoots, shoot length, and number of leaves) pertaining to regeneration of plantlets from leaf calli of Decalepis hamiltonii Wight. & Arn. (swallow root). The variables evaluated were the levels of sucrose, BA, and NAA each at two different concentrations. Response surfaces for shoot length and multiple shoot number were useful in achieving optimal levels of media constituents and in understanding their interactions, but response surfaces for number of leaves were not. The data indicate that sucrose, BA, and NAA levels may be manipulated to increase or decrease quality factors chosen. This approach may be useful in developing a micropropagation protocol for D. hamiltonii. Chemical names used: benzyladenine (BA); napthaleneacetic acid (NAA).

Adventitious shoot regeneration from ovaries of Hosta ‘Golden Scepter’

The objective of this study was to evaluate the ability ofHosta Golden Scepter (GS) ovary explants to generate adventitious shootsin vitro. Ovaries were transversely cut into halves and transferred to petri dishes containingHosta initiation medium supplemented with naphthaleneacetic acid (NAA) at 2.5 μM and N6-benzyladenine (BA) at 10 μM. GS produced adventitious shoots from the ovary base via organogenesis. The number of adventitious shoots regenerated from callus increased linearly with repeated subculturing on Murashige and Skoog (MS) medium supplemented with 2.5 μM NAA and 10 μM BA. The number of multiple shoots developing from callus (15.8), shoot tip (8.4), leaf (6.7), and root (4.3) occurred on MS medium supplemented with 2.5 μM NAA and 20–30 μM BA. There were significant differences in the number of shoots regenerated from shoot tips and callus on MS medium with 50 and 100 mgmyo-inositol per l. Similarly, there were significant differences in the number of axillary shoots and adventitious shoots produced with 20 g/l sucrose treatment.

The Effects of Growth Regulators on Shoot Propagation and Rooting of Vitis Following in Vitro Axillary Bud and Shoot Apex Culture

Axillary buds of `Valiant' grapevine (Vitis spp.) grown in vitro were transferred onto Murashige and Skoog (MS) medium supplemented with different cytokinin and auxin combinations and concentrations. It was found that culture medium caused statistically important differences in number of nodes, number of fully expanded leaves, number of multiple shoots, number of roots, and length of shoots. MS medium supplemented with 1.0 mg BA/liter in combination with 0.01 mg NAA/L was found to be the best medium for shoot growth and callus production. MS medium supplemented with the combination of 0.5 mg BA/L and 0.01 mg NAA/L was the best medium for explant rooting. The medium containing BA and NAA encouraged better shoot growth than those containing BA alone. When the concentration of BA in the medium was increased, multiple shoot proliferation and teratological structures of explants increased, but the number of small leaves and length of internode decreased. Axillary bud culture led to better shoot growth than was found for shoot apex culture. The presence of leaves positively affected shoot growth from axillary buds. Also placing the axillary buds horizontally onto the medium gave better shoot proliferation and growth than placing them vertically.

Effect of liquid culture on the growth and development of miniature rose (Rosa chinensis Jacq. ?Minima?)

The growth of miniature rose (Rosa chinensis Jacq. ‘Minima’) shoots cultured on liquid medium was greater relative to those cultured on two-phase (solid + liquid) medium or solid medium alone. Shoot multiplication ratio (number of multiple shoots per explant per subculture) on liquid medium was higher with 17.8–26.6 μM 6-benzyladenine at compared to that at 0–8.9 μM. Shoots grown on 30 ml or more of liquid medium had a higher multiplication ratio than those grown on 10 or 20 ml. The growth and multiplication ratio increased when the culture period was extended from 3 to 6 weeks, although plantlets began to exhibit some chlorosis by the 6th week. These conditions were maintained over four subcultures for cultivars ‘Baby Katie’, ‘Lavender Jewel’, ‘Red Sunblaze’ and ‘Royal Sunblaze’, with no significant change in multiplication ratio over time.