Single Axillary Bud Research Articles

In vitro conservation of Valeriana officinalis L. through minimal growth

Minimal growth storage could be utilized for in vitro plant conservation. Modifying the culture medium or the environmental condition can reduce the plant growth. The aim of the experiment is to observe the effect of macronutrients on the reduction of valerian culture This research was conducted at Tissue Culture Laboratory at the Indonesian Crops and Medicinal Research Institute (ISMCRI). Single axillary buds with leaves of valerian were used as explants. The treatment applied were :1) full-MS medium + 0.1 mg l-1 BAP, 2) ¾ MS + 0.1 mg l-1 BAP, 3) ½ MS + 0.1 mg l-1 BAP and, 4) ¼ MS + 0.1 mg l-1 BAP. After 12 weeks of conservation, the cultures were transferred into a plant growth regulator free-MS medium to observe their regeneration ability after the conservation. Then, the cultures were acclimatized at the greenhouse to observe their further growth. The result showed that using ¼ MS + 0.1 mg l-1 BAP was the best treatment to reduce the growth during 12 weeks of conservation. After in vitro storage, the cultures can grow normally in full strength plant growth regulator free-MS medium, which was characterized by the formation of new shoots. Valerian also produced roots in the same medium. All acclimatized plantlets were successfully established at the greenhouse.

A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro

‘Candidatus Liberibacter asiaticus (CLas)’, which causes citrus Huanglongbing (HLB) disease, has not been successfully cultured in vitro to date. Here, a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck). Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds. A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination (DAG). This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse. To minimize contamination during in vitro regeneration, CLas-infected axillary buds were micrografted onto seedlings of ‘Changyecheng’ sweet orange and cultured in a liquid medium. In this culture, the titers of CLas in regenerated shoots rapidly increased from 7.5×104 to 1.4×108 cells μg–1 of citrus DNA during the first 40 DAG. The percentages of shoots with >1×108CLas cells μg–1 DNA were 30 and 40% at 30 and 40 DAG, respectively. Direct tissue blot immunoassay (DTBIA) indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse. The disease symptoms in the plantlets were die-back, stunted growth, leaf necrosis/yellowing, and defoliation. The death rate of the plantlets was 82.0% at 60 DAG. Our results show that CLas can effectively multiply in citrus plantlests in vitro. This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

Rapid In Vitro Multiplication of Non-Runnering Fragaria vesca Genotypes from Seedling Shoot Axillary Bud Explants

Fragaria vesca L. has become a model species for genomic studies relevant to important crop plant species in the Rosaceae family, but generating large numbers of plants from non-runner-producing genotypes is slow. To develop a protocol for the rapid generation of plants, leaf explants were compared to single axillary bud shoot explants, both from in vitro-grown Fragaria vesca seedlings, as sources of shoots for new plant production in response to benzyladenine (BA) or thidiazuron (TDZ) combined with indolebutyric acid (IBA) on Murashige and Skoog’s Basal Salt (MS) medium. BA at 2.0 and 4.0 mg L−1 and TDZ at 1.5 mg L−1 promoted the greatest number of shoots produced per shoot explant. There were no IBA effects or IBA interactions with BA or TDZ. Significant interactions between BA and IBA, but not TDZ and IBA, occurred in leaf explant callus formation and % explants with callus at 6 and 9 weeks of culture and on shoots per leaf explant at 9 weeks. TDZ treatments produced uniformly high levels of callus but low numbers of shoots. The treatment generating the most shoot production was BA at 4.0 mg L−1 plus IBA at 0.50 mg L−1. After 9 weeks of culture, leaf explants of the non-runner-producing genotype Baron Solemacher had generated 4.6 shoots per explant with the best treatment, while axillary bud explants had generated 30.8 shoots with the best treatment. Thus, in vitro culture of shoot axillary bud explants can generate high numbers of clonal shoots from a single seedling plant in vitro.

Transcriptome analysis of axillary bud differentiation in a new dual-axillary bud genotype of sugarcane

Each node of the sugarcane stem has a single axillary bud. Therefore, to ensure yield, a large number of cane setts are required for planting, which increases production costs. The dual-axillary bud genotype could provide two buds in a same node so as to reduce the amount of cane setts and ensure enough buds when same sowing planted. To studying the molecular characteristics of this genotype, we performed transcriptome sequencing (RNA-seq) analysis with between the dual-axillary bud genotype and its sister line, the single-axillary bud genotype at the axillary bud differentiation stage. A total of 84.78 Gb clean data were obtained, with an average of 10.90 Gb clean data from each genotype. By aligning to the databases, a total of 50,248 unigenes were functionally annotated. 1646 differentially expressed genes (543 up-regulated and 1103 down-regulated genes) were identified between the two genotypes. The expression of genes involved in the carotenoid pathway including F-box proteins, SCF protein complex, and α/β-hydrolase fold enzymes were down-regulated, 9-cis-epoxycarotenoid dioxygenase was up-regulated, the P450 family exhibited both up- and down- regulated. And genes related to shoot branching, including WRKY transcription factors, the Myb-like DNA-binding domain gene family, and bHLH transcription factors showed both up- and down- regulated, the NAC domain gene family, GRAS gene family, bZIP transcription factors, and R2R3-MYB transcription factors all exhibited down-regulated. Overall, we predict that the dual-axillary bud trait in sugarcane may be related to the significant expression of genes involved in the strigolactone pathway, as well as in genes or transcription factors related to shoot branching.

Evaluation of Horticultural Potential and Standardization of Cultivation Practices in the Less Exploited Wild Species, Gymnostachyum febrifugum (Acanthaceae), Endemic to Southern India

More than 70% of the ornamental plant species grown in Indian gardens are of exotic origin, which directly or indirectly affect the existence of indigenous vegetation of the country. In the exploration to find out the possibilities of the still to be exploited indigenous species in gardens, Gymnostachyum febrifugum Benth. a herbaceous plant endemic to southern Western Ghats of India, is systematically evaluated here for its aesthetic potential. Very poor percentage of seed germination was found the major cultivation constrain. The highly condensed stem restricts vegetative propagation also. A successful vegetative propagation method using single noded stem sections and node splits with single axillary buds is developed. Being vegetative propagation means, it also helps to retain the selected desirable qualities uniformly through generations.

Effect of coconut water and growth regulator supplements on in vitro propagation of Corylus avellana L

Corylus avellana L. represents an economically important crop in the European Union. Several protocols of hazelnut micropropagation have been tested but have not yet provided effective methods to allow large-scale propagation for commercial purposes, due to poor proliferation and yield. The aim of this work was to study the in vitro effects of coconut water (CW) in combinations with gibberellins and cytokinins on proliferation and growth of hazelnut in vitro and the effect of explants orientation on shoot growth. Single axillary buds from mature trees of ‘Tonda Gentile delle Langhe’ cultivar collected from suckers and crown were cultured on a modified DKW semi-solid basal medium enriched with coconut water, BAP, IAA and GA3 in several combinations. The addition of 20% coconut water increased the number of adventitious shoots per explant and the combination of 2mg/L BAP, 0.01mg/L IAA and 0.5mg/L GA3 promoted multiplication and shoots elongation. Shoot multiplication and growth were also affected by explant orientation. The highest rates occurred with sucker explants in the horizontal position.

In vitro germplasm conservation of high Δ9-tetrahydrocannabinol yielding elite clones of Cannabis sativa L. under slow growth conditions

Germplasm conservation of a high Δ9-tetrahydrocannabinol yielding variety of Cannabis sativa L. was attempted using synthetic seed technology and media supplemented with osmotic agents. Explants of nodal segments containing single axillary bud were excised from in vitro proliferated shoot cultures and encapsulated in high-density sodium alginate (230 mM) hardened by 50 mM CaCl2. The ‘encapsulated’ (synthetic seeds) and ‘non-encapsulated’ nodal segments were stored at 5, 15 and 25°C for 8, 16 and 24 weeks and monitored for the re-growth and survival frequency under the tissue culture conditions (16-h photoperiod, 25°C) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.5 μM). ‘Encapsulated’ nodal segments could be stored at low temperature 15°C up to 24 weeks with maximum re-growth ability and survival frequency of 60%. Similar to ‘encapsulated’ cultures, the highest re-growth in ‘non-encapsulated’ cultures was observed in the explants kept at 15°C without osmotic agents. Furthermore, the effect of osmotic agents mannitol and sorbitol (2 and 4% w/v, added individually and in combination to the media at culture room conditions i.e. 25°C) on non-encapsulated shoot cultures was also evaluated. A considerable decrease in re-growth and survival was observed in the cultures treated with osmotic agents. Among the cultures treated with different concentrations of osmotic agents, the highest rate of re-growth and survival was observed at the lowest concentration of 2% sorbitol and 2% mannitol individually added to the media. Well-developed plantlets regenerated from ‘encapsulated’ nodal segments were successfully acclimatized inside the growing room with 90% survival frequency. Gas chromatography-flame ionization detection (GC-FID) was used to compare the chemical profile and the concentration of the different cannabinoids (cannabidiol, cannabichromene, cannabigerol, cannabinol, Δ9-tetrahydrocannabinol and tetrahydrocannabivarin) of the plants grown from ‘encapsulated’ nodal segments to that of the donor plant. The data showed similar cannabinoid profile and insignificant differences in the cannabinoids content between the two types of plants. This study is of high significance since the encapsulation technology would allow the prolonged storage (thus reducing the cost of labor) of high-yielding C. sativa germplasm selected for the isolation of THC, a high-value bulk active pharmaceutic.

A two step method for accelerated mass propagation of Dendrocalamus asper and their evaluation in field

An accelerated protocol for large-scale propagation of Dendrocalamus asper, an edible bamboo, has been described. Seven axillary shoots were induced in vitro from each excised tender node (15-20mm in length) containing single axillary bud when nodal segments were inoculated in semisolid Murashige and Skoog (MS) medium fortified with 5mg/l 6-benzylaminopurine (BAP). Maximum multiple shoot formation (14) was observed when in vitro generated axillary shoots were transferred to liquid MS medium containing 5mg/l BAP and 40mg/l adenine sulphate. A maximum of 93.33% shoots were effectively rooted when transferred to liquid MS medium supplemented with 1mg/l indole-3-biutyric acid (IBA). A simple acclimatization procedure of 55days, primarily in cocopeat for 20days and finally in a blend of sand, soil and farm yard manure (1:1:1 v/v), ensured a very high survival rate within next 35days. After acclimatization, rooted plantlets were further multiplied by splitting of rhizomes, formed in vivo within 90days of growth. After 90 and 180days of acclimatization, plants were successfully transferred to the field and maintained in an unirrigated condition with the initial supplementation of farm yard manure @ 10kg/pit; where around 85% survivability with 25 culms per bush attaining an average height of 4.5m was recorded up to four years.

Micropropagation of Costus speciosus (Koen.) Sm. Using Nodal Segment Culture

Nodal segments of Costus speciosus (Koen.) Sm. containing single axillary buds were cultured on Murashige and Skoog medium (MS medium) supplemented with plant growth regulators for inducing plantlets. For breaking of axillary bud dormancy, nodal segments were cultured on 40-70gl-1 sucrose or 1-13 μM adenine sulphate (AdS) supplemented MS basal medium containing 5 μM 6-benzylaminopurine (BAP) and 1μM α-naphthalene acetic acid (NAA). The nodal segments cultured on 1-13 μM AdS, 5 μM BAP, 1 μM NAA and 50gl-1 sucrose showed simultaneous production of shoots and roots while those cultured on 5 μM BAP, 1 μM NAA and 40-70gl-1 sucrose produced shoots only. The most effective media for breaking axillary bud dormancy was 5 μM BAP, 1 μM NAA, 50 gl-1 sucrose and 10 μM AdS supplemented medium. The propagules from 40-70gl-1 sucrose produced roots in shoot multiplication medium, i.e.,10 μM AdS, 1 μM NAA, 50gl-1 sucrose and 3-11 μM BAP supplemented medium. The best response for shoot multiplication was on 10 μM AdS, 1 μM NAA, 50gl-1 sucrose and 7 μM BAP. The well-rooted shoots were hardened and transferred to the soil where they showed 95% survival rate. Results show that axillary bud can be used for micropropagation of Costus speciosus.

In Vitro Propagation of Arundinaria callosa Munro- an Edible Bamboo from Nodal Explants of Mature Plants

A procedure for in vitro propagation of Arundinaria callosa Munro has been developed. The method allows bud-break from nodal segments containing single axillary bud on Murashige and Skoog (1962) medium supplemented with different concentrations of 6- benzylaminopurine (BAP). The early bud-break was obtained in 8.9-13.3 � M BAP, within 8-15 days. The position of the node on the culm of lateral branches also affected bud-break percentage and multiplication, mid-culm nodes are the most suitable. The optimal concentration of 13.3 � M BAP is found significant for shoot multiplication. Addition of 1.0 � M 3- indolebutyric acid (IBA) enhances the shoot multiplication rate. In vitro rooting was induced when 15 � M IBA was incorporated for three subcultures in the shoot proliferation medium, was transferred tostrength MS containing 25� M IBA and 0.05 � M BAP, and finally withdrawn from the rooting medium. The regenerants were successfully transplanted into a soil mixture for acclimatization before field planting.

Micropropagation of Field‐Grown Perennial Teosinte from Node Culture

Propagation of perennial diploid teosinte, Zea diploperennis Iltis, Doebley and Guzmán (2n = 2x = 20), and tetraploid teosinte, Z. perennis (Hitchc.) Reeves and Magelsdorf (2n = 4x = 40), is limited by seed availability. For plants grown in the field, a photoperiod response delays flowering until fall, and seeds may not reach maturity. The objective of this study was to develop an in vitro micropropagation protocol for field grown teosinte. Shoot proliferation was induced from nodes split longitudinally and plated on Murashige and Skoog's (MS) medium supplemented with 5 μM 6‐benzyl amino purine and 3% (w/v) sucrose. Both species possess a single axillary bud that generated multiple plants on division. Rooting of shoots was achieved in half‐strength MS medium with and without the addition of 0.4 μM indole‐3‐butyric acid and 2% (w/v) sucrose. Nodes and younger branches of the tetraploid generated significantly more plants than those of the diploid.

Micropropagation of Vitis vinifera L: towards an improved protocol

Nodal explants bearing a single axillary bud, from three cultivars of cultivated grape, viz., ‘Thompson seedless’, ‘Sonaka’ and ‘Tas-e-Ganesh’, were used to initiate shoot cultures on G16 medium containing adenine sulphate, monobasic sodium phosphate, BAP and NAA. Each shoot, developed from an axillary bud, produced a tuft of multiple shoots on a medium containing BAP, calcium pantothenate, monobasic sodium phosphate and IBA. Subculture of the tuft of multiple shoots to an ‘elongation medium’ resulted in distinct individual shoots. Rooting of shoots and plantlet formation was achieved on IAA-containing liquid medium. A culture procedure for enhanced multiple shoot production and the recovery of complete plants is described. The use of this protocol for the commercial exploitation of cultivated grape is discussed.

In vitro multiplication ofVanilla planifolia using axillary bud explants.

A clonal propagation method has been developed for efficient multiplication ofVanilla planifolia. Multiple shoots were developed from axillary bud explants using semi-solid Murashige and Skoog (MS) medium supplemented with N6-benzyladenine (BA, 2 mg l-1) and α-naphthaleneacetic acid (NAA, 1 mg l-1). The multiple shoots were transferred to agitated liquid MS medium with BA at 1 mg l-1 and NAA at 0.5 mg l-1 for 2-3 weeks, and subsequently cultured on semi-solid medium. Using this method, an average of 42 shoots were obtained from a single axillary bud explant over a period of 134 days. Use of an intervening liquid medium has been found to enhance multiplication of shoots inV. planifolia.

Occurrence of multiple shoots bearing flowers arising from single axillary buds on kiwifruit canes treated with hydrogen cyanamide

Abstract Dormant first‐order axillary buds on kiwifruit (Actinidia deliciosa (A. Chev) C.F. Liang et A.R. Ferguson) canes break in spring and normally produce a single shoot bearing flowers through the extension of the primary bud axis. Documented here is the occurrence of three shoots arising from a single axillary bud, with each shoot bearing flowers, after treatment with hydrogen cyanamide. Given that dormant first‐order buds contain second‐order axillary buds, the multiple shoots reported here are likely to be in part, the result of the extension of the second‐order axillary bud axes. This shows that the axillary meristems found within the second‐order buds also possess the inherent ability to produce flowers.

NODE COUNTING IN AXILLARY BUDS OF NICOTIANA TABACUM CV. WISCONSIN 38, A DAY‐NEUTRAL PLANT

A mature, quiescent, primary axillary bud on the main axis of a flowering Nicotiana tabacum cv. Wisconsin 38 plant, when released from apical dominance and before forming its terminal flower, produced a number of nodes which was dependent upon its position on the main axis. Each bud produced about one more node than the next bud above it. The total number of nodes produced by an axillary bud was about 6 to 8 greater than the number of nodes present above this bud on the main axis. At anthesis of the terminal flower on the main axis, mature, quiescent, primary axillary buds had initiated 7 to 9 leaf primordia while secondary axillary buds, sometimes present in addition to the primary ones, had initiated 4 to 5 leaf primordia. When permitted to grow out independently, primary and secondary axillary buds located at the same node on the main axis produced the same number of nodes before forming their terminal flowers. In contrast, immature primary axillary buds which had produced only 5 leaf primordia and which were released from apical dominance prior to the formation of flowers on the main axis produced only as many nodes as would be produced above them on the main axis by the terminal meristem, i.e., “extra” nodes were not produced. Therefore, it is the physiological status of the plant and not the number of nodes on the bud at the time of release from apical dominance that influenced the node‐counting process of a bud. When two axillary buds were permitted to develop on the same main axis, each produced the same number of nodes as single axillary buds developing at these nodes. Thus, the counting process in an axillary bud of tobacco is independent of other buds. Axillary buds on main axes of plants that had been placed horizontally produced the same number of nodes as identically‐positioned axillary buds on vertical plants, indicating that gravity does not play a major role in the counting, by an axillary bud, of the nodes on the main axis.

Node Counting in Axillary Buds of Nicotiana tabacum cv. Wisconsin 38, a Day-Neutral Plant

A mature, quiescent, primary axillary bud on the main axis of a flowering Nicotiana tabacum cv. Wisconsin 38 plant, when released from apical dominance and before forming its terminal flower, produced a number of nodes which was dependent upon its position on the main axis. Each bud produced about one more node than the next bud above it. The total number of nodes produced by an axillary bud was about 6 to 8 greater than the number of nodes present above this bud on the main axis. At anthesis of the terminal flower on the main axis, mature, quiescent, primary axillary buds had initiated 7 to 9 leaf primordia while secondary axillary buds, sometimes present in addition to the primary ones, had initiated 4 to 5 leaf primordia. When permitted to grow out independently, primary and secondary axillary buds located at the same node on the main axis produced the same number of nodes before forming their terminal flowers. In contrast, immature primary axillary buds which had produced only 5 leaf primordia and which were released from apical dominance prior to the formation of flowers on the main axis produced only as many nodes as would be produced above them on the main axis by the terminal meristem, i.e., “extra” nodes were not produced. Therefore, it is the physiological status of the plant and not the number of nodes on the bud at the time of release from apical dominance that influenced the node-counting process of a bud. When two axillary buds were permitted to develop on the same main axis, each produced the same number of nodes as single axillary buds developing at these nodes. Thus, the counting process in an axillary bud of tobacco is independent of other buds. Axillary buds on main axes of plants that had been placed horizontally produced the same number of nodes as identically-positioned axillary buds on vertical plants, indicating that gravity does not play a major role in the counting, by an axillary bud, of the nodes on the main axis.

CONTROL OF SHOOT‐RHIZOME DIMORPHISM IN THE WOODY MONOCOTYLEDON, CORDYLINE (AGAVACEAE)

In Cordyline terminalis negatively geotropic leafy shoots and positively geotropic rhizomes develop from single axillary buds on either shoots or rhizomes. All axillary buds have similar morphogenetic potential when released from apical dominance. Experiments in which the orientation of the apex is changed, organs removed, or growth regulators applied indicate that after a rhizome is initiated, it is maintained as a rhizome by auxin originating in the leafy shoot. When auxin levels are lowered by changes in the orientation of the axis or shoot removal, the rhizome apex becomes a shoot apex, which appears to be the stable state of the actively growing apex. Benzyl adenine when applied exogenously to the apex or lateral buds has the same effect as lowering the auxin level. Gibberellic acid has no effect on the apex or lateral buds. High levels of exogenous naphthaleneacetic acid cause bud release and development of rhizomes from previously inhibited axillary buds of the shoot. However, it was not possible to convert a shoot apex into a rhizome apex by auxin treatment. It is suggested that the release of buds on the lower side of horizontal branches and of buds directly above a stem girdle is caused by high auxin levels on the lower side or distal to the girdle. The experimental results are discussed in relation to naturally occurring shoot‐rhizome dimorphism.

Control of Shoot-Rhizome Dimorphism in the Woody Monocotyledon, Cordyline (Agavaceae)

In Cordyline terminalis negatively geotropic leafy shoots and positively geotropic rhizomes develop from single axillary buds on either shoots or rhizomes. All axillary buds have similar morphogenetic potential when released from apical dominance. Experiments in which the orientation of the apex is changed, organs removed, or growth regulators applied indicate that after a rhizome is initiated, it is maintained as a rhizome by auxin originating in the leafy shoot. When auxin levels are lowered by changes in the orientation of the axis or shoot removal, the rhizome apex becomes a shoot apex, which appears to be the stable state of the actively growing apex. Benzyl adenine when applied exogenously to the apex or lateral buds has the same effect as lowering the auxin level. Gibberellic acid has no effect on the apex or lateral buds. High levels of exogenous naphthaleneacetic acid cause bud release and development of rhizomes from previously inhibited axillary buds of the shoot. However, it was not possible to convert a shoot apex into a rhizome apex by auxin treatment. It is suggested that the release of buds on the lower side of horizontal branches and of buds directly above a stem girdle is caused by high auxin levels on the lower side or distal to the girdle. The experimental results are discussed in relation to naturally occurring shoot-rhizome dimorphism.