Frequency Of Shoot Bud Regeneration Research Articles

An efficient in vitro shoot regeneration protocol from embryo explants of chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is an important grain legume in the world. Development of reliable tissue culture protocols is very important for genetic transformation studies. Direct shoot bud differentiation and multiple shoot induction from embryonal axes explants of chickpea varieties with combination of growth regulators in NBeG-3 and NBeG-72 was tried at Regional Agricultural Research Station, Nandyal in 2015-16. Explants were cultured on Murashige and Skoog (MS) medium augmented with different concentrations of Benzyl amino purine (BAP), Napthalene acetic acid (NAA), Kinetin and Indole acetic acid (IAA). The frequency of shoot bud regeneration was influenced by the preconditioning of explants with 6mg/l BAP and 0.5mg/l thidiazuron (TDZ) for 10 days resulted in enhanced frequency of multiple shoots. Among the various concentrations tested, 4.0 mg/l BAP and 2.0 mg/l Napthalene acetic acid (NAA) were found to be the best for maximum shoot bud differentiation. Elongation of multiple shoots were obtained in MS medium with the concentration 0.5 mg/l gibberillic acid (GA3). Among the tested varieties there was a variation in regeneration ability with fastest shoot bud initiation (18.5 days), and highest number of shoots per explant (5.3) and highest shoot length (3.5 cm) in NBeG 3.

High frequency plant regeneration from cotyledonary node explants of Cucumis sativus L. cultivar ‘Green Long’ via adventitious shoot organogenesis and assessment of genetic fidelity by RAPD-PCR technology

Influence of cytokinins, silver nitrate (AgNO3) and auxins on plant regeneration from cucumber was investigated. The cotyledonary node explants were cultured on MS medium augmented with various concentrations (0.5-2.5mg l-1) of 6-benzyl amino purine (BAP) and kinetin (KIN) for shoot bud induction. BAP at 1.5mg l-1 was found to be the best concentration for induction of high frequency of multiple shoots (98.4%). Interestingly, maximum percent of multiple shoot regeneration (100%) as well as number of shoot buds (54.6 shoots/culture) was recorded on MS medium containing the combination of 4.5mg l-1 AgNO3 and 1.5mg l-1 BAP. Multiple shoot bud regeneration frequency as well as the number of shoots was positively correlated with the concentrations of AgNO3. Addition of silver nitrate in the medium not only enhanced the rate of multiple shoot bud regeneration but also elongation of shoot buds was observed. The highest percent of rooting (96.2%) was noticed on a medium containing the combination of indole 3-butyric acid (IBA), 1.5mg l-1 and KIN 0.5mg l-1. Acclimatized plantlets were successfully established in the field where the survival rate observed was 72%. The RAPD profiles of in vitro regenerated plants were found to be highly monomorphic and identical banding pattern with mother plant. DNA fingerprinting results confirmed that the tissue culture plantlets were found to be true-to-type. The present study describes efficient protocol for high frequency plant regeneration via adventitious shoot organogenesis in cucumber.

Direct shoot regeneration from immature inflorescence cultures of Chlorophytum arundinaceum and Chlorophytum borivilianum

Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L−1 BA, 150 mg L−1 Ads, 0.1 mg L−1 NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation. Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L−1 IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement prograrmme.

Effects of gibberellin mutations on shoot bud regeneration of Arabidopsis

Tissue culture provides a useful system to investigate how plant hormones areinvolved in this process. Auxin and cytokinin are widely used in plant regeneration. Gibberellin is also an important plant hormone in regulating plant growth and development. It is interesting to know the effects of gibberellin and its signalling pathway on plant regeneration. In this report Arabidopsis thaliana landsberg (wild type), ga1-3 (gibberellin biosynthesis deficiency mutant), gai (gibberellin insensitive mutant), penta mutant (lacking GA1, GAI, RGA, RGL1, RGL2) and tetra mutant (lacking GAI, RGA, RGL1, RGL2) were used as materials to investigate how plant regeneration progress was affected in these mutants under different conditions.The results showed that more shoot buds were regenerated in ga1-3 and gai than in wild type, penta and tetra mutant in the normal shoot induction medium. The frequency of shoot bud regeneration in different mutants also varied remarkablywhen auxin : cytokinin ratio in the medium changed. Only penta mutant had shoot bud regeneration without auxin in the medium. When the ratio increased wild type,ga1-3 and gai had higher performance in shoot bud regeneration than penta mutant.When cytokinin level increased from 0.1 to 0.5 mg l-1, shoot bud regeneration frequency of ga1-3, wild type and tetra increased remarkably except for that of pentaand gai. Only the shoot bud regeneration frequency of gai did not change significantly when cytokinin level increased from 0.5 to 5 mg l-1. These gibberellin-related mutants also responded differently to NPA (an auxin polar transport inhibitor) in plant regeneration. Our results indicate gibberellin and its related pathway are also involved in plant organogenesis: gibberellin inhibitor and auxin polar transport inhibitor can promote plant organogenesis. This might provide a new way for the regeneration of recalcitrant species. Key words: Shoot bud regeneration, Arabidopsis thaliana, gibberellin, DELLA protein mutant.

Effects of gibberellin mutations on in vitro shoot bud regeneration of Arabidopsis

Tissue culture provides a useful system to investigate how plant hormones are involved in this process. Auxin and cytokinin are widely used in plant regeneration. Gibberellin is also an important plant hormone in regulating plant growth and development. It is interesting to know the effects of gibberellin and its signalling pathway on plant regeneration. In this report Arabidopsis thaliana landsberg (wild type), ga1-3 (gibberellin biosynthesis deficiency mutant), gai (gibberellin insensitive mutant), penta mutant (lacking GA1, GAI, RGA, RGL1, RGL2) and tetra mutant (lacking GAI, RGA, RGL1, RGL2) were used as materials to investigate how plant regeneration progress was affected in these mutants under different conditions. The results showed that more shoot buds were regenerated in ga1-3 and gai than in wild type, penta and tetra mutant in the normal shoot induction medium. The frequency of shoot bud regeneration in different mutants also varied remarkably when auxin : cytokinin ratio in the medium changed. Only penta mutant had shoot bud regeneration without auxin in the medium. When the ratio increased wild type, ga1-3 and gai had higher performance in shoot bud regeneration than penta mutant. When cytokinin level increased from 0.1 to 0.5 mg l-1, shoot bud regeneration frequency of ga1-3, wild type and tetra increased remarkably except for that of penta and gai. Only the shoot bud regeneration frequency of gai did not change significantly when cytokinin level increased from 0.5 to 5 mg l-1. These gibberellin-related mutants also responded differently to NPA (an auxin polar transport inhibitor) in plant regeneration. Our results indicate gibberellin and its related pathway are also involved in plant organogenesis: gibberellin inhibitor and auxin polar transport inhibitor can promote plant organogenesis. This might provide a new way for the regeneration of recalcitrant species.

High-frequency induction of multiple shoots and clonal propagation from rhizomatous nodal segments of Houttuynia cordata Thunb.—An ethnomedicinal herb of India

This study reports an efficient and direct shoot bud differentiation and multiple shoot induction from nodal segments of underground stoloniferous rhizomes of Houttuynia cordata Thumb. The frequency of shoot bud regeneration was influenced by the type of cytokinin and concentrations. Among the various concentrations used, benzylaminopurine (BAP, 17.74 μM) or kinetin (Kn, 18.58 μM) was found to be most effective for rapid and maximum shoot but differentiation. The number of shoots per explant was higher (20.00±2.61) on Murashige and Skoog (MS) medium supplemented with Kn (18.58 μM) compared to BAP and 6-γ-γ-(dimethyl-allylamino)-purine (2iP) during initial 40-d-old culture. Subsequent shoot differentiation and multiplication were achieved in MS medium containing 9.29 μM Kn and 15% (v/v) coconut milk. Elongation and growth of multiple shoots were also obtained on MS medium containing either 2.32 μM Kn or 2.46 μM 2iP alone. The rate of shoot multiplication during subcultures declined with an increase in the size of proliferating shoot cluster. Reducing shoot cluster size to three to four shoots and subculturing together in shoot multiplication medium resulted in a better shoot multiplication and growth, which could be maintained for 2 yr. The elongated shoots (>20 mm) were successfully rooted on MS medium supplemented with 19.60 μM indole-3-butyric acid. Regenerated plants were successfully established in soil and were found to be healthy and uniform. The protocol reported in this study can be used for conservation and utilization of elite clone of H. cordata.

Direct plant regeneration of curry leaf tree (Murraya koenigii koenig.), an aromatic plant

An efficient protocol for plant regeneration from stem segments of Murraya koenigii was developed by culturing on Murashige and Skoog (MS) medium supplemented with 2.5 mg l−1 benzyladenine (BA), 25 mgl−1 adenine sulfate, 0.25 mgl−1 indole-3-acetic acid (IAA), and 3% sucrose. The frequency of shoot bud regeneration was higher on similar medium in subsequent subcultures. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.25–0.5 mgl−1 IAA or 1-naphthaleneacetic acid (NAA) within 8–12 d of culture. The maximum percentage of rooting was obtained on MS medium supplemented with IAA and NAA, each at 0.25 mgl−1. During acclimatization, 95% of rooted plantlets survived were grown normally under greenhouse conditions.

Phytochrome B Influences in-vitro Shoot Regeneration in Arabidopsis thaliana (L.) Heynh.

The effects of phyA and phyB mutations on shoot bud regeneration in Arabidopsis thaliana have been studied. The phyB mutant showed a significantly lower frequency of shoot bud regeneration than did the wild-type, indicating a role for phytochrome B in the regulation of in-vitro shoot bud regeneration. Interestingly, the shoot bud regeneration frequency of the phyA mutant was not significantly different from that of the wild-type in a light and dark, suggesting that phytochrome A may not be involved in the regulation of in-vitro shoot bud regeneration. The effects of gibberellin (GA) were also studied because previously we showed that a reduction in endogenous GA level results in a higher shoot bud regeneration frequency. Exogenous paclobutrazol and prohexadione (GA biosynthesis inhibitors) increases, and exogenous GA reduces, the shoot bud regeneration frequency in the phyB mutant. Thus shoot regeneration in the phyB mutant is still responsive to regulation via the GA signalling system.

Rapid and High-Frequency In Vitro Plant Regeneration from Leaflet and Petiole Explants of Groundnut ( Arachis hypogaea L.)

An efficient protocol for regeneration of groundnut plantlets from immature and mature leaflet and petiole explants excised from axenic seedlings has been developed. The highest frequency of callus induction obtained from leaflet explants was on Murashige and Skoog (MS) medium containing 2.0 mg/L of α-naphthalene acetic acid (NAA) and 0.5 mg/L of kinetin combination. MS medium containing different auxins in combination with 6-benzylaminopurine (BAP) induced shoot buds. A BAP (2.0 mg/L) and NAA (0.5mg/L) combination resulted in the highest frequency of shoot-bud regeneration. Subsequent shoot multiplication was obtained on MS medium supplemented with either BAP or kinetin (5.0 mg/L) in combination with NAA (1.0 mg/L). Immature leaflet explants were found to be more responsive to shoot induction than mature leaflet explants. Direct shoot-bud regeneration was also observed from petiole explants on the same regeneration medium used for leaflet callus. Regenerated shoots were rooted on MS medium containing indole-3-butyric acid (2.0 mg/L) and kinetin (0.5 mg/L). Plantlets obtained were successfully established in the field, where they grew to maturity and set viable seeds.

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l−1 (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N6-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l−1 (4.4 μM). NAA (0.1 mg.l−1, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l−1, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l−1 (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.

In vitro plant regeneration via callogenesis and organogenesis in Bambusa vulgaris

Friable calli were induced from mature excised shoots of Bambusa vulgaris on Murashige and Skoog's (MS) medium supplemented with 2.2 µM6-benzylamino-purine (BAP), 9.04 µM 2,4-dichlorophenoxyacetic acid and 14.76 µM indole-3-butyric acid (IBA) with 3 % (m/v) saccharose. Adventitious shoots with root hairs were achieved from calli on MS medium supplemented with 13.33 µM BAP and 1.23 - 2.46 µM IBA within 4 weeks of subculture. The frequency of shoot bud regeneration was better in the light incubated cultures than in the dark incubated cultures. Isolated shoots were rooted on liquid half-strength MS basal medium supplemented with 0.49 µM IBA and 2 % (m/v) saccharose. Histological observations confirmed the regeneration of shoot buds from calli. The rooted plantlets were successfully transferred to greenhouse.

Effect of auxins and cytokinins on efficient plant regeneration and multiple-shoot formation from cotyledons and cotyledonary-node expiants of groundnut (Arachis hypogaea L.) by in vitro culture technology

Tissue cultures were established from cotyledon and cotyledonarynode segments ofArachis hypogaea L. on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins (IAA, NAA, IBA, and 2, 4-D) and cytokinins (KIN and BAP). For callus initiation, high concentration of auxins and low concentration of cytokinins were used, whereas high concentration of cytokinins and low concentration of auxins were used for shoot-bud differentiation. Callus induction and shoot-bud regeneration frequency, however, varied with genotype, expiant, and the different plant-growth regulators combination in the medium. The shoot-bud multiplication was also influenced by genotype, explant type, and growth regulators. The combination of BAP and NAA produced more shoots than other combinations. The maximum number of shoots was obtained from cotyledonary-node segments on a medium containing BAP (5.0 mg/L) and IBA (1.0 mg/L). Rooting of regenerated shoots was achieved on a medium augmented with NAA or IBA (2.0 mg/L) in combination with KIN (0.5 mg/L). Rooted plantlets were successfully established in the soil, where 95% of them survived.

Endogenous gibberellin levels influence in-vitro shoot regeneration in Arabidopsis thaliana (L.) Heynh.

The regeneration of shoot buds from callus cells in vitro is an important technique in modern plant genetic manipulation. Whilst it is clear that genetic factors play a major role in determining the ability of callus cells to become organized into regenerating shoot buds, the precise nature of these factors remains unknown. Here we show that callus derived from mutants of Arabidopsis thaliana which have reduced levels of endogenous bioactive gibberellins (GAs), or reduced responsivity to GAs, regenerates shoot buds more readily than does callus derived from wild-type controls. In addition, exogenous GA reduces, and exogenous paclobutrazol (a GA-bio-synthesis inhibitor) increases, the frequency of shoot bud regeneration from wild-type callus. These results show that GA levels play a role in regulating shoot bud regeneration from callus, and suggest that variation in endogenous GA levels or responsivity may account for a major component of the genetic variation in shoot bud regeneration frequency described in other species.

Tuberous Roots an Ideal System for High Frequency In vitro Regeneration in Plumbago rosea L.

Tuberous root explants of Plumbago rosea L. cultured in MS containing 3.5 mg/l BAP showed high frequency of shoot bud regeneration (62.33 ± 0.66; 0.26 ± 0.07 cm) within eight weeks. Subculture of shoot clumps (22.17 ± 0.90 shoots; 0.26 ± 0.07 cm) from the initial clump exhibited extensive rate of shoot multiplication (123.4 ± 1.029 shoots, 0.22 ± 0.01 cm) per each clump and produced a total of 369 shoots in 3.5 mg/l BAP in six weeks. Within two weeks of culture in basal liquid medium, shoot elongation (6.16 ± 0.14 cm) occurred and after three weeks in MS solid medium supplemented with 0.1 mg/l IAA, rooting (14.7 ± 1.2) initiated on the shoots. Ninety six per cent of the rooted plants were established under the shade nethouse with regular watering. Key words: Plumbago rosea, tuberous root, multiple shoots, High frequency D.O.I. 10.3329/ptcb.v20i2.6915 Plant Tissue Cult. & Biotech. 20(2): 203-209, 2010 (December)