μM Indole-3-butyric Acid Research Articles

THE INFLUENCE OF THIDIAZURON ON IN VITRO SHOOT PROLIFERATION OF OAKLEAF HYDRANGEA (HYDRANGEA QUERCIFOLIA BARTR.)

The objectives of this study were to promote adventitious shoots from leaf explants and axillary shoots from nodes in vitro. Leaf explants were excised from 12-15 month old shoot cultures and the nodal explants were taken from greenhouse-grown stock plants. The leaf explants were obtained by cutting leaves that formed in vitro in half and placing them on MS medium with 1 μM indolebutyric acid (IBA) plus thidiazuron (TDZ) at 5x10 -8 , 1x10 -7 , 5x10 -7 , 1x10 -6 , or 5x10 -6 M. The explants were transferred to fresh media every 28 days and the shoot number and shoot length data were taken at the end of 16 weeks. The concentration of TDZ in the medium significantly affected adventitious shoot number with the most shoots forming on leaf explants on medium with 5x10 M TDZ. Although a mean of 68.7 shoots formed with this concentration of TDZ, the majority were < 0.5 cm long. As the concentration of TDZ decreased, the number of adventitious shoots that formed were significantly fewer, but longer. Nodal and terminal bud explants from greenhouse-grown plants were placed horizontally on MS media with 1 μM IBA plus 10 -10 , 10 -9 , 10 -8 , 10 -7 , or 10 -6 M TDZ. The nodal explants were transferred to fresh media every 28 days and data were taken at 9 weeks. Total shoot production was greatest at 10 -6 M TDZ, with a mean of 5.2 shoots. At this concentration, there was a combination of axillary and adventitious shoots but as the concentration of TDZ decreased there were predominately axillary shoots.

In vitro propagation of Acacia sinuata (Lour.) Merr. from nodal segments of a 10-year-old tree

An in vitro propagation protocol has been developed using nodal explants from a mature ‘elite’ tree of Acacia sinuata. Tissue browning was circumvented by soaking surface-disinfected explants in a solution of antioxidant (238 μM citric acid). Maximum shoot proliferation (75.2%) was achieved from nodal explants collected during the December to March season in Murashige and Skoog's (MS) medium supplemented with 8.9μM 6-benzyladenine (BA), 2.5μM thidiazuron (TDZ), and 135.7μM adenine sulfate (AS) at the end of the first transfer following initial culture (60 d after inoculation). Gibberellic acid (GA3) at 1.8 μM promoted shoot elongation. The number of shoots was increased by (1) repeated subculturing of nodal explants on fresh medium with the same composition, and (2) using microcuttings from in vitro-regenerated shoots on MS medium containing 6.6 μM BA where each node produced four shoots. When transferred to half-strength MS medium augmented with 7.4 μM indolebutyric acid (IBA) in vitro-regenerated shoots produced prominent roots. Rooted plants were hardened and successfully established in soil. This protocol yielded an average of 100 plants per nodal explant over a period of 3 mo.

Assessing competence for adventitious shoot formation in hypocotyl explant cultures fromCatharanthus roseus cultivars

Hypocotyl expiants from 22 cultivars ofCatharanthus roseus were cultured on various shoot-inducing media to assess their competence for adventitious shoot formation. The Murashige and Skoog (MS) media had been supplemented with 14 μM zeatin and 2.5 μM indole-3-butyric acid (IBA), 4.5 μM BA and 0.5 μM α-naphthaleneacetic acid (NAA), or 14 μM thidiazuron and 2.5 μM IBA. After eight weeks, the expiants from ‘Cooler Raspberry Red’ showed the greatest frequency of adventitious shoot formation, followed by ‘Cooler Orchid’ and ‘Cooler Treated’. The highest frequency (86.7%) for ‘Cooler Raspberry Red’ was attained on the medium enhanced with 14 μM zeatin and 2.5 μM NAA. Excised adventitious shoots were then readily rooted on a half-strength MS basal medium. Afterward, the regenerated plantlets were transferred to potting soil and grown to maturity in a greenhouse.

Lateral root development and saponin accumulation as affected by IBA or NAA in adventitious root cultures of Panax ginseng C.A. Meyer

The effect of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) on lateral root formation was investigated in adventitious root culture of Panax ginseng. Lateral root formation was affected by IBA (24.6 μM) or NAA (9.8 μM). Lateral root primordia emerged from the explant root pericycle after about 7 d of culture when the roots were cultured on Schenk and Hildebrandt (SH) medium supplemented with 24.6 μM IBA or 9.8 μM NAA. However, no changes were observed in the explant root pericycle on auxin-free medium. The IBA treatment was more effective for lateral root induction and root growth compared to NAA. In morphological and histological aspects, the lateral roots formed under IBA treatment developed normally, while NAA-treated roots exhibited abnormal growth. The accumulation of total saponin was greater in roots treated with IBA than with NAA.

Efficient plant regeneration in Holarrhena antidysenterica Wall., from shoot segment-derived callus

A procedure has been outlined for plant regeneration of an important medicinal shrub, Holarrhena antidysenterica, through shoot segment-derived callus. Explants used for callus induction were shoot segments derived from 14-d-old axenic plants on Murashige and Skoog (MS) medium supplemented with 15 μM N6-benzyladenine (BA). A white friable type of callus was obtained in 4.52 μM 2,4-dichlorophenoxyacetic acid and 2.32 μM kinetin which did not have the potentiality to regenerate. High-frequency shoot differentiation was achieved on transferring the friable callus to MS medium supplemented with 17.8 μM BA and 8.0 μM naphthaleneacetic acid. The highest percentage of calluses forming shoots (65.06±2.26) was achieved in this medium. The organogenetic potential of the regenerating callus was influenced by the age of the culture. Rooting was achieved on the shoots using MS medium with 25 μM indolebutyric acid. The plantlets were acclimatized and established in soil. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.

Plant Regeneration from Mesophyll Protoplasts of Goodenia scaevolina F. Muell.

To establish a protoplast culture system of Goodenia scaevolina F. Muell., cotyledonary mesophyll protoplasts were isolated and cultured. Because seeds of G. scaevolina are difficult to germinate, the smoke medium was employed to stimulate their germination to obtain cotyledons. After the pre-treatment of cotyledons in a liquid medium, consisting 1/2 strength of MS supplemented with 0.6 M sucrose at 25°C in the dark for 1-2 hr, they were transferred to a solution containing 1 % Cellulase Onozuka RS, 0.1 % Pectolyase Y-23, 0.6 M mannitol and protoplast washing salts and kept at 25°C in the dark for 15 hr. Protoplasts were cultured in 1/2 MS liquid medium supplemented with 1 μM 2, 4-dichlorophenoxyacetic acid, 0.1 μM, 6-benzylaminopurine (BAP) and 0, 6 M sucrose. Using the sucrose for an osmoticum and a carbon source enabled protoplasts to grow up to callus in the initial medium without any additional treatment. When the calli grew to 1-2 mm in diameter, they were transferred onto 1/2 MS solid medium, supplemented with 0.1 μM indole-3-butyric acid (IBA), and 10 μM BAP for plant regeneration. After rooting on MS medium supplemented with 2 μM IBA, the regenerated plantlets were acclimatized and subsequently flowered.

Rapid in vitro propagation of medicinally important Ceropegia candelabrum

Protocol for rapid in vitro propagation of Ceropegia candelabrum L. (Asclepiadaceae) through axillary bud multiplication was established. Murashige and Skoog (MS) medium with 8.87 μM N 6-benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA) was best suited for axillary bud proliferation inducing a mean of eight shoots / node. Excision and culture of the nodal segments from the in vitro shoots on fresh medium with same concentrations of BA and IBA facilitated development of more than 10 shoots / node. Subsequent cultures enhanced the rate of shoot proliferation. Shoots developed were rooted best on half strength MS with 0.49 μM IBA. Starting from a single node explant, 250 rooted shoots were obtained within 120 days. Plantlets established in pots exhibited 90% survival. Plantlets successfully established in field exhibited morphological characters identical to mother plants.

Plant regeneration from excised hypocotyl explants of Platanus acerifolia willd

A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l−1 (8.88 μM) BA and 0.5 mg l−1 (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.

Effective Protocol for in Vitro Shoot Production Through Nodal Explants of Simmondsia Chinensis

Nodal explants excised from 18 to 20-year-old female plants of Simmondsia chinensis if cultured on Murashige and Skoog's medium supplemented with 20 μM N6-benzyladenine (BA) differentiated an average of 2.7 ± 0.4 shoots in 11.5% explants. The percentage of nodal explants inducing multiple shoots enhanced significantly if in vitro raised shoots were used as source of explants. Nearly 100% cultures differentiated an average of 4.7 ± 2.0 shoots per explant on the same medium. Nearly 85% of the shoots induced roots when a pulse treatment of 50 μM indole-3-butyric acid (IBA) was given prior to their transfer to semi-solid MS medium containing 10 μM IBA + 0.5% activated charcoal + 1 μM BA. Plantlets were gradually hardened in Soilrite and acclimatized to soil.

Gelling agents, silver nitrate and sequestrene iron influence adventitious shoot and callus formation from Rubus leaves

Blackberry (Rubus spp.) cultivars Chester Thornless, Kotata, Marion, and Navaho were evaluated for morphogenic potential using the top one to four leaves from in vitro-grown shoots. In each experiment adventitious shoots were regenerated via organogenesis without an intervening callus phase. Rooting was spontaneous when shoots were transferred to a medium without growth regulators or with indole-3-butyric acid (IBA). Three gelling treatments (agar, Gelrite, and a combination) calibrated for equal gel firmness [110 g/(1.1 cm)2 π] did not affect explant regeneration or the number of shoots per explant, but did affect callus production. Significantly more callus (P≤0.05) was induced on regeneration medium (RM) with 10 μM N6-benzyladenine (BA), 0.5 μM IBA, and 0.19% Gelrite, than on medium containing either 0.71% agar, or the 0.35% agar/0.11% Gelrite combination. Explants on RM with 5 μM IBA produced significantly more callus, but fewer shoots, compared to zero or 0.5 μM IBA treatments for all gel treatments. Adding 200 mg l−1 sequestrene iron [sodium ferric ethylene diamine di-(O-hydroxyphenylacetate)] at the first transfer onto RM induced more shoots per explant than the control, but did not influence the amount of callus produced. Sequestrene iron in the second transfer on RM significantly increased the regeneration (caulogenesis) frequency from 30 to 40% for ‘Marion’ and from 23 to 43% for ‘Kotata’. Silver nitrate significantly reduced callus production for all treatments, but did not affect the frequency of caulogenesis or the number of shoots per explant.

An Improved Protocol for Regeneration of Sorghum bicolor from Isolated Shoot Apices.

An improved and producible protocol for in vitro regeneration of sorghum [Sorghum bicolor (L.) Moench] from shoot meristem explant is reported. By striking an optimal balance between a weak auxin like naphthalene acetic acid (NAA) and the cytokinin thidiazuron (TDZ), the isolated shoot meristems were manipulated to follow either organogenic or embryogenic pathway. There was no intermediate callus formation. Multiple buds were induced on enlarged meristems using MS medium with 5.0μM of TDZ, 17.72μM Benzylaminopurine (BAP), and 1.074μM NAA. To maximize the number of bud initials per explant, three parameters (seed size, germination technique, and age of explant) were studied. Five to 7-day-old shoot meristems responded best with ≥80% induction of bud initials. Seed size was not significant in influencing induction potential of the shoot meristems. Six weeks after in vitro culture, each meristem produced 35-40 shoot buds. Direct somatic embryogenesis (≥80% induction) was accomplished following a two-step culture procedure consisting of induction of multiple buds and formation of somatic embryos. A high frequency (700-1000) of somatic embryos per explant was obtained on MS medium with 17.72μM BAP and 2.69 μM NAA. Shoots from both organogenic and embryogenic pathways rooted on half-strength Murashige and Skoog (MS) medium with 8.28μM Indole 3-butyric acid (IBA) and 1.14μM Indole acetic acid (IAA). After one month on rooting medium, plants with well-developed roots were transferred to jiffy cups. Such plants were subsequently acclimatized in the glasshouse, and were grown till maturity; they showed normal seed set. Random amplified polymorphic DNA (RAPD) analysis of regenerants did not detect any DNA polymorphism.

In vitro micropropagation of Piper longum: an important medicinal plant

Efficient and rapid tissue culture systems were developed for Piper longum, an important medicinal plant, through shoot tip multiplication and direct regeneration. Multiple shoots were induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.44–22.19 μM benzyladenine (BA) and 4.64–13.9 μM kinetin (K). Maximum number of shoots were induced with 8.9 μM BA and 4.64 μM K. Adventitious shoot regeneration from leaf segments was achieved on MS containing 3.6–22.19 μM BA along with 3.31–12.4 μM picloram (P). Shoot differentiation occurred directly from the leaf bases without intermediale callus formation. Maximum shoot buds were obtained on MS medium with 17.76 μM BA and 8.28 μM P. Elongated shoots were separated and rooted in MS supplemented with 2.46 μM indole butyric acid (IBA). Plantlets, thus developed were established in soil.

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Acacia sinuata is a valuable multipurpose tree in Southern India. The tree is over exploited, but its regeneration rate in natural habitat is low. Therefore, it is important to study if it can be regenerated through in vitro micro-propagation. Cotyledonary node and shoot-tip explants excised from 15 day-old in vitro grown seedlings were used to initiate cultures. Maximum number of shoots was induced from cotyledonary node explants on Murashige and Skoog's (MS) medium containing6.66 µM 6-benzylamino purine (BAP) and 4.65µM kinetin (Kn). Subculturing was done in the fresh medium of same composition. The number of shoots formed was comparatively greater in the first subculture. Maximum shoot elongation was achieved (5.5 cm)when subcultured on MS medium supplemented with 1.75 µMgibberellic acid (GA3). In vitro regenerated shoots produced roots when transferred to half strength MS medium supplemented with 7.36 µM indolebutyric acid (IBA). From each cotyledonarynode 30 shoots were obtained within 90 days after two subcultures. The success rate of establishing the rooted plantlets in the field was 55%.

In vitro propagation of Iphigenia indica, an alternative source of colchicine

The present study involves in vitro propagation of Iphigenia indica (Kunth.) through multiplication of whole corms and corm buds. The whole corms produced very small micro-corms, which developed plants individually whereas corm buds multiplied to produce numerous shoots at variable rates in presence of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). The best response in corm and bud multiplication was obtained in Murashige and Skoog's basal medium (MS) supplemented with 2.69 μM NAA and 8.88 μM BAP. The shoots regenerated were further cultured on MS medium containing NAA and indole-3-butyric acid (IBA) for initiation of roots. MS medium with 5.38 μM NAA and 4.92 μM IBA induced highest percentage of roots (81%) within 2 weeks in culture.

In vitro propagation of the Greek olive cultivar `Chondrolia Chalkidikis'

The effect of basal media (Woody Plant Medium (WPM), Quoirin and Lepoivre (QL) and Olive Medium (OM)) and of various concentrations of cytokinins (6-benzyladenine (BA), zeatin, 6-(γ,γ-dimethylallyl-amino) purine (2iP)), solely or in combinations with each other and with gibberellic acid (GA3), on in vitro shoot proliferation of the greek olive cultivar `Chondrolia Chalkidikis' was investigated. WPM proved to be the most effective one, resulting in better morphological appearance of the microshoots produced. The highest number of new microshoots/explant (1.68), with a 3.0 cm shoot height and a 4.2 proliferation rate, was obtained when this medium was supplemented with 20 μM zeatin. The number of microshoots/explant and proliferation rate increased to 1.85 and 6.8, respectively, by using the combination of 5–20 μM zeatin with 1 μM BA, but shoot height was reduced. 2iP was the least effective of the cytokinins tested. Combination of 20 μM zeatin with 10 μM GA3 affected positively shoot proliferation resulting in 1.80 microshoots/explant, 3.0 cm shoot height and 7.0 proliferation rate. However, the same concentration of GA3 in combination with 1 μM BA reduced the number of new microshoots/explant (0.48) as well as the proliferation rate (2.4), although shoot height remained nearly the same (2.7). The effect of indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and putrescine on root induction was also studied. A rooting percentage of up to 70% and 2.3 roots/microshoot were achieved by the combination of 12 μM IBA + 3 μM NAA, however, abscission of shoot tips and leaves appeared. Both variables were increased, up to 93% and 4.0, respectively, by the addition of 30 μM putrescine in the medium, without any undesired side effect. After acclimatization, survival of rooted microshoots was high (90%) while that of non-rooted ones was low (30%).

Large-scale production of Anogeissus pendula and A. Latifolia by micropropagation

Success has been achieved in developing a complete protocol for mass propagation of Anogeissus pendula and A. latifolia, two important forest species found in India. Seeds cultured on plant growth regulator-free, semisolid Murashige and Skoog (MS) medium germinated within 5–6 wk and formed 4–6-cm long shoots. The shoots multiplied on MS+4.4 μM benzyladenine (BA)+5.7 μM indoleacetic acid (IAA) + casein hydrolysate (100 mgl−1) + ascorbic acid (50 mgl−1) + sucrose (3%) + agar (0.8%). A majority of the genotypes rooted with more than 90% efficiency when 5–6 cm individual shoots were cultured on 1/2MS (only major salts reduced to half strength)+2.3 μM IAA+2.5 μM indolebutyric acid (IBA) + sucrose (3%)+agar (0.8%) for 15 d. Those 10% (approx.) genotypes that did not root well on the above medium could be rooted with ease by increasing the concentration of IAA in the rooting media from 2.3 to 5.7 μM. The in vitro-raised plants were successfully transferred to the soil with a success rate of over 85%. Using this protocol, over 560 000 tissue-cultured plants of these two species have been produced and dispatched to various state forest departments for field trials and routine plantations.

Micropropagation of Allium wallichii kunth, a threatened medicinal plant of Nepal

Bulbs and aerial parts of the Nepalese plant Allium wallichii are widely used for medicinal purposes and as a spice. Due to overharvesting the natural populations of the species have been increasingly reduced and the domestication of the species should be considered. For the purpose of the production of plantlets suitable for field culture, a micropropagation procedure based on multiple shoot culture has been established. Multiplication factors of 4.6 on average were possible on MS medium supplemented with 20 μM zeatin. After rooting on MS medium with 10 μM indolebutyric acid, plantlets were acclimatized to greenhouse conditions and transferred to the field with good success.

Micropropagation of Rollinia mucosa (JACQ.) baill

A protocol for in vitro propagation of Rollinia mucosa, an important medicinal plant, was developed. The presence of 500 mg l−1 polyvinylpyrrolidone (PVP) during explant excision was important to avoid browning. Axillary buds, adventitious buds, and shoot cluster proliferation were achieved from epicotyl and hypocotyl explants from nursery-grown seedlings. The highest direct organogenesis percentage from hypocotyl explants was obtained upon culture of explants on Murashige and Skoog medium supplemented with 2.2 μM benzyladenine (BA) plus 2.32 μM kinetin. Epicotyl explants display highest regeneration frequency on a medium containing 8.8 μM BA and 0.54 μM naphthaleneacetic acid. Gibberellic acid was necessary for shoot elongation. Root induction was observed when shoots were pretreated with activated charcoal for 7 d in the dark before culture on Woody Plant Medium supplemented with 49.21 μM indolebutyric acid for 10 d. Root development was observed when 20 g l−1 sucrose was used. Rooted plantlets were acclimatized and grown in the greenhouse.

Somatic embryogenesis and plant regeneration in Medicago arborea L.

An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 μM) and kinetin (9 μM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 μM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 μM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18.0±0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets (8.0±0.89%) when cultured on basal MS medium with 5 μM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N6-benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 μM.

In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.