Axillary Shoot Induction Research Articles

In Vitro Shoot Culture of Sesuvium portulacastrum: An Important Plant for Phytoremediation

Sesuvium portulacastrum L., a member of the family Aizoaceae, is an important coastal halophyte. Due to its adaptability to salinity and heavy metals, S. portulacastrum has now been widely used for the phytoremediation of saline soils and wastewater and the protection of the coast from erosion. The increasing use of this plant requires a large number of propagules. Stem cutting propagation and seed germination cannot meet this demand, and such propagations can initiate and spread diseases. A recent occurrence of Bipolaris sesuvii J.Z. Zhang and Gibbago trianthemae E.G. Simmons in S. portulacastrum resulted in the substantial loss of the plants during the remediation of aquaculture wastewater. Thus, there is an urgent need for establishing efficient methods of propagating disease-free starting materials. In the present study, we evaluated different growth regulators in the induction of axillary shoots from nodal explants cultured on Murashige and Skoog medium and identified that zeatin (ZT) and α-naphthaleneacetic acid (NAA) was an appropriate combination for inducing high numbers of axillary shoots. The nodal explants were then cultured on MS medium supplemented with different concentrations of ZT and NAA, and the combination of ZT at 1.0 mg L−1 and NAA at 0.3 mg L−1 induced more than 12 axillary shoots per explant. The axillary shoots were excised to produce microcuttings or microshoots, which were rooted on half-strength MS medium supplemented with different concentrations of indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The results showed that IBA at 0.6 mg L−1 induced 91.7% of the microcuttings to root with root numbers of over 36 per cutting. The rooted plantlets were healthy and true-to-type and grew vigorously in plug trays or plastic containers with a 100% survey rate in a greenhouse. Thus, this established protocol could be used for the rapid propagation of genetically identical and disease-free plants of S. portulacastrum for phytoremediation and the protection of shoreline soils from erosion.

In vitro shoot culture of Rhododendron fortunei: An important plant for bioactive phytochemicals

Plants in the genus Rhododendron L. produce more than 200 compounds and have been considered an important source for bioactive phytochemicals. Production of these plants, however, requires healthy and genetically uniform propagules. This study evaluated effects of culture media and growth regulators on axillary shoot induction of R. fortunei Lindl. as well as auxin for in vitro and ex vitro rooting of microcuttings. Protocols for in vitro shoot culture of this species were developed. Two-node explants were cultured on either Economou and Read (ER) medium or woody plant medium (WPM) supplemented with 4.0 mg L−1 6-(4-hydroxy-3-methylbut-2-enylamino) purine or zeatin (ZT) and 1.0 mg L−1 naphthaleneacetic acid (NAA). More than 10 axillary shoots were produced per explant regardless of medium. The axillary shoots were used to produce additional two-node explants or as microcuttings for rooting. Two-node explants derived from the axillary shoots cultured on the same media produced even higher numbers of shoots, ranging from 17 to 20 on both ER medium and WPM from the second to the third or fourth cycle of culture, shoot numbers started decreasing in the fourth or fifth cycle. Microcuttings were easily rooted in vitro with rooting percentages of 100% on ER and 84.0% on WPM supplemented with 1.0 mg L−1 indole-3-butyric acid (IBA). The rooting percentages were almost 100% in a peat-based substrate irrespective IBA treatment. Micropropagated plants were morphologically stable and grew vigorously with a 95% survival rate in a greenhouse. The established methods could be used for rapid propagation of disease-free and genetically uniform liners which can be used to produce feedstocks for extracting bioactive phytochemicals.

Role of Phytohormones and PGR's in Micropropagation of Anethum graveolens L. through Axillary Shoots and Evaluation of NaCl effect on Growth Parameters

An In vitro protocol is standardized for rapid in vitro propagation of Anethum graveolens through enhanced axillary shoot induction and optimization of role of BAP on shoot proliferation. MS basal medium augmented with various hormones (BA, NAA, KIN, and IBA) was used for multiple shoot development and rhizogenic response. The influence of various concentrations of NaCl was also observed on plant growth and morphological parameters. MS media fortified with BA (0.5–1.5) singly was induce axillary bud proliferation and in combination with NAA (0.1–0.25 mg L−1) and KIN (0.25–0.50 mg L−1), it was able to induce multiple shoots. BA at 1.0 mg L−1, KIN 0.5 mg L−1 and NAA 0.25 mg L−1 induced optimum number of multiple shoots (11.00 ± 0.379). The maximum frequency of rooting 83% with 3.67 ± 0.66 roots per explants was recorded on half strength MS medium supplemented with 1/2 MS + IBA 1.0 mg L−1 within 4 weeks of transfer of the shoots to the rooting medium. Hardening was successfully attained under partially controlled conditions inside the hardening room. The method could effectively used for conservation and clonal propagation to meet the pharmaceutical demands of this medicinally important plant species.

Micropropagation of blueberry ‘Bluejay’ and ‘Pink Lemonade’ through in vitro shoot culture

Abstract This study systematically evaluated effects of culture media, cytokinins, and auxins on axillary shoot induction as well as auxins and media or substrates on in vitro and ex vitro rooting of a highbush cultivar Bluejay (Vaccinium corymbosum L.) and a V. ashei rabbiteye hybrid derivative ‘Pink Lemonade’. Optimal protocols for in vitro shoot culture of the two cultivars were developed. Two-node stem explants derived from axillary shoots were cultured on Anderson medium supplemented with 13.68 μM 6-(4-hydroxy-3-methylbut-2-enylamino) purine (ZT) and 0.27 μM naphthaleneacetic acid (NAA) for inducing axillary shoots of ‘Bluejay’ and 9.12 μM ZT and 0.05 μM NAA for ‘Pink Lemonade’ shoot proliferation. Microcuttings derived from the axillary shoots were cultured on half-strength Anderson medium supplemented with 0.49 μM indole-3-butyric acid (IBA) for in vitro rooting. The rooting percentages for ‘Bluejay’ and ‘Pink Lemonade’ were 75% and 65% in two months, respectively. However, by dipping microcuttings into 4.92 μM IBA for 10 s and rooting ex vitro in a substrate composed of 90% peat and 10% perlite, the rooting percentages could be improved to 89% for ‘Bluejay’ and 97% for ‘Pink Lemonade’. After being acclimatized in a shaded greenhouse for three months, rooted plants were ready for planting in the field. The established methods could be used for rapid propagation of highbush and rabbiteye hybrid derivative cultivars for commercial blueberry production.

Micropropagation of Weigela florida ‘Tango’ through In Vitro Shoot Culture

Weigela florida (Bunge) A. DC. is a popular flowering shrub adapted to a wide range of environmental conditions. Efficient methods for micropropagation of this species have not been well developed. The present study established a protocol for in vitro shoot culture of W. florida ‘Tango’ after a systematic evaluation of different culture media, cytokinins, and auxins on axillary shoot induction. Single-node stems were cultured on Driver and Kuniyuki Walnut (DKW) medium for initial production of axillary shoots. The shoots were used as explants and cultured on DKW medium supplemented with 8.88 μm 6-benzylaminopurine (BA) and 0.27 μm naphthaleneacetic acid (NAA), resulting in the production of more than six axillary shoots per explant. The axillary shoots could either be used as explants for additional shoot production or be cultured on ½ DKW medium supplemented with 0.25 μm indole-3-butyric acid (IBA) for rooting. Plantlets were transplanted into a substrate with 99% survival rate in a shaded greenhouse. This established method could be used for rapid propagation of W. florida to speed the introduction of new hybrids or cultivars for commercial production.

High-efficiency Propagation of Mature ‘Washington Navel’ Orange and Juvenile ‘Carrizo’ Citrange Using Axillary Shoot Proliferation

In vitro axillary shoot proliferation can be used to increase availability of citrus (Citrus) types in high demand, while limiting somaclonal variation. However, established protocols could be improved to increase efficiency. Therefore, this study investigated some factors [plant growth regulators (PGRs), basal media, and successive subculturing] which affect the in vitro axillary shoot proliferation of mature ‘Washington Navel’ orange (Citrus sinensis) and juvenile ‘Carrizo’ citrange (C. sinensis × Poncirus trifoliata). In ‘Washington Navel’ orange, maximum axillary shoot induction (66.9% explants producing axillary shoots with a mean of 2.45 shoots per explant) was obtained in Driver and Kuniyuki walnut (DKW) medium supplemented with 0.1 mg·L−1 6-benzylaminopurine (BA), 0.05 mg·L−1 naphthalene acetic acid (NAA) along with 1 mg·L−1 6-furfurylaminopurine [kinetin (kin)], whereas in ‘Carrizo’ citrange, axillary shoot production was greatest (82.6% and 87.5% of explants producing axillary shoots with a mean of 4.3 and 4.1 shoots per explant) at 1.0 or 2.0 mg·L−1 BA in DKW medium. The initial nodal propagules (with basal tissue remaining from removed shoots) were repeatedly subcultured for six times every 4 weeks onto DKW medium with the same levels of PGRs used for initial culturing. Woody plant medium (WPM), Murashige and Skoog medium (MS), and DKW were also compared for rooting at quarter to full strength for salt components, all amended with 2.0 mg·L−1 indolebutyric acid (IBA) and 0.5 mg·L−1 NAA. MS at full strength provided the highest rooting in ‘Carrizo’ citrange (93%) and longest root length (58 mm), whereas half-strength MS provided the highest rooting in ‘Washington Navel’ orange (60% to 61%) and the longest roots (26 mm). Addition of 1 μm spermidine to the rooting medium enhanced root length only for ‘Washington Navel’ orange on full-strength MS, but accelerated rooting for both cultivars on all media. The plantlets were successfully transferred to greenhouse conditions, exhibiting normal development, with high uniformity, and no evidence of somaclonal variation.

In vitro propagation and anthraquinone quantification in Gynochthodes umbellata (L.) Razafim. & B. Bremer (Rubiaceae)—A dye yielding plant

In vitro propagation techniques have been standardized for axillary shoot multiplication through nodal and shoot tip explant for the first time in Gynochthodes umbellata, a natural dye yielding plant. Single nodes and shoot tip obtained from young top shoots were cultured on Murashige and Skoog agar medium (MS) fortified with different concentrations of different cytokinins like BA, KIN, 2ip and TDZ and its combinations. The positions of nodes from the young shoots have some influence in the percentage axillary shoot induction and multiple shoot formation. Of the different position of the nodes used, the 4th node from the shoot tip showed maximum percentage (85%) response and multiple shoot formation in presence of BA. Of the different cytokinine used, BA had a dominant role in promoting multiple shoot proliferation in the nodal explant. An average of 6.8270±0.28 shoots/node was obtained in MS medium containing 2mg/l BA. The optimal medium for rooting was half strength MS medium (in dark) fortified with 1.5mg/l IAA (17.20±7.55 roots/shoot) with basal callusing, where 1.5mg/l NAA gave maximum of roots (7.90±3.25 roots/shoot) without any basal callus formation and showed highest establishment rate in community pots as well as in the field (90%).The roots of one year old established in vitro derived plants showed higher amount of anthraquinone (8.330mg/g fw) than the in vivo plants (4.567mg/g fw). The described protocol can be effectively used for the large scale propagation, exploitation of active compound and conservation of this important dye yielding plant.

In vitro propagation and preliminary results of Agrobacterium-mediated genetic transformation of Cordyline fruticosa

A method for in vitro propagation through axillary shoot proliferation and induction of adventitious shoots as well as Agrobacterium-mediated transformation of Cordyline fruticosa is presented. In vitro Cordyline shoots were cultured on MS medium supplemented 0–4mgl−1 thidiazuron (TDZ) for their multiplication. MS medium supplemented with 0.1mgl−1 TDZ proved to be the most effective in which 14 shoots per explants were obtained. Leaf segments were excised from in vitro axillary shoots and cultured on MS medium supplemented with TDZ (0–5mgl−1) for callus induction. Following induction, the non-embryogenic calluses were proliferated on MS medium without plant growth regulators. A rapid and efficient method for producing transgenic plants has been developed for Cordyline via Agrobacterium-mediated transformation. Leaf segments from in vitro shoots were co-cultivated with Agrobacterium tumefaciens EHA105, which harbored the binary vector carrying the bar and β-glucuronidase (GUS) genes in the T-DNA region. The results showed that the number of plants expressing GUS gene first increased with inoculation time and bacterial density (OD600), and then dramatically decreased with the increment of both factors. The highest percentage of transformation efficiency (85%) was obtained when the leaf segment explants were inoculated with Agrobacterium for up to 40min at OD600 of 1.0 followed by 80 and 75% at OD600 of 0.8 and 1.2, respectively. The transgenes were confirmed in transgenic plants using GUS primers and gave the expected band size of 750bp. The results suggested that infection time and Agrobacterium density could have some effects on the transformation efficiency. Southern blot analysis was used for definitive confirmation of tDNA integration in Cordyline and confirmed the results of histochemical GUS assay and PCR amplifications. Regenerated plantlets were acclimatized in greenhouse.

Induction and proliferation of axillary shoots from in vitro culture of Paeonia lactiflora Pall. mature zygotic embryos

Zygotic embryos of three herbaceous peony (Paeonia lactiflora Pall.) cultivars (‘Fen Yu Nu’, ‘Zhong Sheng Fen’ and ‘Zhu Sha Pan’) were used to study embryo germination and optimum plant growth regulator (PGR) combinations for in vitro propagation and root initiation. Mature zygotic embryos (>90 days after flowering [DAF]) gave better germination and survival than immature zygotic embryos (50–70 DAF). A protocol for initiating shoot growth and axillary shoot proliferation was established using mature embryos. The best results were obtained when using excised zygotic embryos (EZEs) obtained by removing the testa and endosperm from the seed. The best medium for EZE germination (an embryo with cotyledons) was half-strength Murashige and Skoog (MS) medium (with double-strength CaCl2) supplemented with 0.5 mg L−1 6-benzyladenine (BA) and 0.5 mg L−1 gibberellic acid (GA3). The best medium for axillary shoot proliferation was half-strength MS medium (including double-strength CaCl2) supplemented with 1 mg L−1 BA and 1 mg L−1 GA3. PGR-free half-strength MS was the best medium for promoting root development on seedlings (a germinated EZE with tender leaves) and for robust in vitro seedling establishment. The acclimatization of herbaceous peony remains the most challenging step of the in vitro protocol.

Short Communication. An efficient axillary shoots propagate system for the living fossil plant - Wollemi pine (Wollemia nobilis)

Aim of study: The aim of the work was to determine the optimum axillary shoot induction system by optimizing different factors.Area of study: The different factors which were pretreatment methods, media types, kinds of cytokinins and dark treatmentMaterial and methods: After pretreating and sterilizing, the stems of Wollemi pine which about 3cm long were cultured on three kinds of media (GD, DCR AND ms) with different concentration and type of cytokines (BA, ZT and TDZ), as well as dark treatment (0, and 4 weeks) and then exposure to tissue culture room. The experiments were randomized block designed and the results were analyzed with ANOVA.Main results: Rapid freezing in liquid nitrogen was the most effective pretreatment method, dark treatment for the first 4-weeks of stem explants cultured in induction media could increase the frequency of axillary shoot induction significantly. The highest frequency of axillary shoot induction (63%) was obtained when explants were cultured in 1/2GD medium with 6.8 μM TDZ combining 4-weeks dark treatment. Research highlights: The optimized conditions for shoot production in vitro can be useful for conservation in vitro of Wollemia nobilis.Keywords: Wollemia nobilis; in vitro conservation; tissue culture; an endangered relict conifer.

The influence of medium solidifying agents and double-phase medium on the growth and development of Cosmos atrosanguineus (Hook.) Voss in vitro

Influence of different medium gelling agents (Agar-Agar Sigma, Lab-AgarTM Biocorp, Bacto-Agar Difco, Gelrite) and two types of medium (solidified by Agar-Agar and double-phase medium) on branching and growth of Cosmos atrosanguineus shoots was investigated. Shoot tips obtained from aseptical tissue cultures were grown for 6 weeks on Murashige and Skoog medium containing BA in concentration of 1 mg ∙ dm-3. It was found that induction of axillary shoots was the best on double-phase medium (addition of liquid medium after 4 weeks of shoot cultures in vitro). No significant differences were found in regeneration potential and elongation of shoots depending of the media solidified by studied agars or Gelrite.

Stable Agrobacterium-mediated transformation of maritime pine based on kanamycin selection.

An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.

Effects of culture conditions on multiple shoot induction from inflorescence and RAPD analysis of cloned plants in Limonium sinensis (Girard) Kuntze, var. Golden Diamond

The optimum physical and chemical microenvironment for micropropagation of Limonium sinensis (Girard) Kuntze, var. Golden Diamond was established from immature inflorescence segments as explant. The highest frequency (62 %) of axillary shoot induction was obtained on MS medium (Murashige and Skoog Physiol Plant 15:473–497, 1962) supplemented with 8.88 μM BA, 1.34 μM of NAA and two growth additives cysteine (142.33 μM), and glutamine (684.22 μM). In the subsequent culture maximum average number of shoots (11.13 ± 0.34) were obtained from micro-shoot explant on MS medium supplemented with the same additives and 2.22 μM BA. During subcultures the problem of vitrification was mitigated through increasing agar concentration from 0.8 % to 1.0 % and providing better ventilation. The in vitro developed shoots were rooted on MS medium supplemented with 2.46 μM IBA and 0.88 μM BA. Rooted plants were acclimatized successfully in the greenhouse with 80 % survival rate. RAPD analysis using 15 random decamer primers generated monomorphic banding pattern in micropropagated plants and similar to those of mother plant revealing the genetic integrity of regenerants.

Multiple shoot induction and rooting of Paeonia lactiflora ‘Da Fu Gui’

Underground buds of herbaceous peony (Paeonia lactiflora Pall.) ‘Da Fu Gui’ weremicropropagated in vitro. The basic processes including culture initiation, shoot induction, axillary shoot proliferation and rooting were established. The best initial medium of ‘Da Fu Gui’ was half-strength MS (Murashige and Skoog) medium (double-strength Ca2+) supplemented with 0.5 mg l-1 6-benzylaminopurine (BA) plus 0.5mg l-1gibberellic acid (GA3). The best medium for axillary shoot induction was half-strength MS medium (double-strength Ca2+) supplemented with 1.0 mg l-1 BA plus 0.5 mg l-1kinetin (Kin), while 0.5 mg l-1 BA + 0.3 mg l-1 Kin was best for shoot proliferation. Shoot height at the time of inoculation had a great effect on proliferation and growth of ‘Da Fu Gui’. Putrescine (Put) (0.5 to 5.0 mg l-1) prevented rooting of ‘Da Fu Gui’ but it favored the development of roots. Highest rooting percentage was observed on half-strength MS medium (double-strength Ca2+) supplemented with 1 mg l-1 indole-3-butyric acid (IBA). Key words: Herbaceous peony, underground buds, axillary shoots, micropropagation.

Direct shoot induction of Paeonia lactiflora ‘Zhong Sheng Fen’ and rejuvenation of hyperhydric shoots

The underground buds of herbaceous peony (Paeonia lactiflora Pall.) ‘Zhong Sheng Fen’ were used to investigate the effects of indole-3-acetic acid (IAA) on culture initiation, benzyladenine (BA) on axillary shoot induction, and three auxins (IAA, indole-3-butyric acid [IBA] and 1-naphthyleneacetic acid [NAA]) on axillary shoot proliferation. In addition, methods to rejuvenate hyperhydric microshoots were established. Our results showed that the best initiation medium for ‘Zhong Sheng Fen’ was half-strength Murashige and Skoog (MS) medium supplemented with double the concentration of Ca2 + (880 mg L−1), 1 mg L−1 BA, 0.5 mg L−1 gibberellic acid (GA3) and 0.1 mg L−1 IAA. Axillary shoots were successfully induced by 2.0 mg L−1 BA. Several methods allowed the successful rejuvenation of hyperhydric microshoots, allowing them to develop normally. These included the addition of 3 g L−1 activated charcoal, the removal of ammonium nitrate from the medium, doubling the concentration of Ca2 + or eliminating BA from the medium.

Improvement of shoot morphogenesis in vitro and assessment of changes of the activity of antioxidant enzymes during acclimation of micropropagated plants of Desert Teak

The aim of the study was to develop an improved and rapid regeneration system via axillary shoot proliferation for Tecomella undulata, an important endangered medicinal tree in India. The Murashige and Skoog (MS) medium augmented with different concentrations (0.1, 0.3, 0.5, 0.7, 1.0, 2.5, 5.0, 7.5 and 10.0 μM) of Thidiazuron (TDZ) was tested for the ability to induce axillary shoot development from cotyledonary node explants excised from 7-day-old sterile seedlings. Among the tried concentrations of TDZ, 0.7 μM showed optimum response in inducing maximum number (25.00 ± 2.30) of shoots and shoot length (4.06 ± 0.58 cm) after 3 weeks of incubation. The regenerated shoots when subcultured on MS medium lacking TDZ gave twofold shoot multiplication rate with maximum number (43.00 ± 2.86) of shoots per explant and longer shoot length (7.40 ± 0.34 cm) during the fourth subculture passage. Attempts were also made to study the morphogenetic effect of medium pH and sucrose concentration on axillary shoot induction and proliferation. The highest efficiency of shoot regeneration was recorded in MS medium supplemented with 0.7 μM TDZ and 3% sucrose at pH 5.8 after 3 weeks of culture. Different concentrations of Indole-3-butyric acid (IBA) were tested to determine the optimum conditions for ex vitro rooting of microshoots. The best result was accomplished with IBA (200 μM) pulse treatment given to the basal end of the microshoots for 30 min followed by their transfer in plastic cups containing soilrite and eventually established in natural soil with 80% survival rate. During acclimatization of plants, catalase (CAT) activity increased reaching maximum at 28th day after transplantation, while superoxide dismutase (SOD) activity increased reaching a maximum in the 21 days. Likewise, changes in the glutathione reductase (GR) and Ascorbate peroxidase (APX) were also detected. The observed changes reflect the plant’s capacity to develop antioxidant mechanisms during acclimatization which determine the ability to survive in oxidative stress. The standardized protocol for mass propagation of Tecomella undulata should eliminate the dependence on natural stands of plants for pharmaceutical purposes, and will also serve as a means of conservation as the species is highly overexploited.

An efficient in vitro process for recurrent production of cloned plants of Vitex negundo L

An efficient method for cultivation of Vitex negundo L. through axillary shoot (collected from micropropagated plants) proliferation has been successfully developed, which can be employed at a commercial scale. Axillary shoot induction was most successful using nodal explants for propagation on Murashige and Skoog (MS) medium supplemented with various concentrations of single cytokinin or in various combination with auxins. We obtained the maximum percentage (97.6 ± 1.45) response with highest number (16.4 ± 0.60) of shoots per explant on MS medium supplemented with 6-benzyladenine (BA) and α-naphthalene acetic acid (NAA) at concentrations of 5.0 and 0.5 μM, respectively. Shoot regeneration frequency was optimized by manipulating pH and using various media. MS medium and pH 5.8 was found to be the optimum for maximum regeneration. Nodal explants from in vitro regenerated microshoots too developed shoots, thus making the process recurrent. Shootlets with 4–5 nodes were utilized for in vitro rooting, and best response was evaluated on MS medium supplemented with 1.0 μM indole-3-butyric acid (IBA). The well-developed micropropagated plants were acclimatized (97%) successfully within 2 weeks in soilrite and planted ex vitro in normal garden soil, where they grew well without any morphological and genetic variations. The present regeneration process not only favoured the rapid multiplication but also expressed the regeneration capability of micropropagated plants.

GENOTYPIC DIFFERENCES IN SHOOT MULTIPLICATION AMONG FIVE CITRUS ROOTSTOCKS IN VITRO

The present study aims at developing optimal media for different cultivars of Citrus namely, 'Rangpur' lime, 'C 35' citrange, 'Troyer' citrange, 'Swingle' citrumelo and 'Alemow' macrophylla. Cultures from adult field-grown trees were established from nodal stem segments for axillary proliferation. The single node segments were surface-sterilized and cultured on Murashige and Skoog (MS medium; 1962) medium. The shoot cultures obtained were used as explants to investigate the effects of various media constituents necessary for growth. Among the media constituents, cytokinins played a crucial role in shoot proliferation and thus were a prerequisite for the shoot multiplication media in all the cultivars. In case of 'Alemow' macrophylla, a synergistic effect of auxins and cytokinins was observed. The effect of sucrose concentration was also variety-dependent and significantly affected the production of axillary shoots, with either 3% or 4% being the optimal concentration depending upon the rootstock. Moreover, the influence of gelling agents also varied with each cultivar. For 'Rangpur' lime, gelrite (0.2%) was the optimum gelling agent whereas agar-gel (0.4%) was the best for the remainder four varieties. Among the five rootstocks tested, shoot proliferation rate was highest in 'Rangpur' lime and lowest in 'Alemow' macrophylla. The results demonstrate that genotypic differences played a significant role in the induction and growth of axillary shoots. Thus, in this study we were able to identify and optimize the most effective multiplication media for each cultivar, which is a necessary step to develop an efficient micropropagation protocol.

Effect of Urea—Type Citokinins on Axillary Shoots Regeneration of Impatiens Walleriana L.

ABSTRACTTo establish an efficient procedure for in vitro plant regeneration of Impatiens walleriana L., the effects of urea-type cytokinins, forchlorofenuron (CPPU) and thidiazuron (TDZ) on the axillary shoot induction are examined. The shoot meristem explants produced multiple axillary shoots when cultured on Murashige and Skoog's (MS) medium containing different concentration of CPPU and TDZ (0.01–1.0 μM). Explant activity varied significantly depending on the cytokinin type as well as concentration. We have found that the 0.1 μM CPPU induced the largest number of axillary shoots (4.89). This cytokinin was more effective for the induction and development of axillary shoots than TDZ in the same concentration. The separated shoots are rooted and elongated on MS medium without hormone. Rooted plantlets were successfully acclimatized. Shoot meristem explants and urea-type cytokinins could be useful for improvement of regeneration procedure and genetic engineering of this important ornamental species.

Effect of riboflavin on initiation and multiplication of shoot cultures of sugarcane

Early induction of axillary shoots in sugarcane has been obtained by using MS medium supplemented with riboflavin 10 mg/l along with growth regulators BAP (0.25 mg/l), NAA (0.5 mg/l) and kinetin (1.07 mg/l). Lowest level of riboflavin (1 mg/l) with the combination of 2.0% sucrose induced higher frequency of multiplication.