Maximum Number Of Multiple Shoots Research Articles

Enhanced shoot multiplication in Ficus religiosa L. in the presence of adenine sulphate, glutamine and phloroglucinol.

Ficus religiosa (Pipal) is a long-lived valuable multipurpose forest tree. The tree is exploited because of its religious, ornamental and medicinal value and the regeneration rate in natural habitat is low. An in vitro propagation protocol has been developed from nodal segments obtained from a 45-50-year old tree. The highest bud break frequency (100 %) followed by maximum number of multiple shoots (13.9) as well as length (2.47cm) were obtained on Woody Plant medium (WPM) supplemented with 1.0mg/l BAP along with 0.5mg/l IAA. Two modifications in this medium resulted in enhanced shoot regeneration-one with 200mg/l glutamine + 150mg/l ADS (called as MM-1) giving 32.5 shoots per nodal explant while another modification-with 200mg/l glutamine + 150mg/l ADS + 100mg/l phloroglucinol (called as MM-2) giving 35.65 shoots per explant. These two media were used for sub-culturing of shoots for 4months. The rate of shoot multiplication was same during the first three sub-cultures on MM-1 and the shoots regenerated were healthy, afterwards shoot multiplication declined. While on MM-2, shoot multiplication declined after first sub-culture and shoots underwent the problem of early leaf fall. Rooting was best induced in micro-shoots excised from proliferated shoot cultures on semi-solid as well as liquid WPM modified with 2.0mg/l IBA and 0.5mg/l IAA. The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 25-30days before transfer to soil conditions, where the established plants showed more than 90 % survival.

MULTIPLE SHOOT REGENERATION FROM THE CULTURES OF ADHATODA VASICA NEES.

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

Direct shoot organogenesis on hypocotyl explants with collar region from in vitro seedlings of Coffea canephora Pierre ex. Frohner cv. C × R and Agrobacterium tumefaciens-mediated transformation

Direct differentiation of shoot buds from the collar region of hypocotyl segments of Coffea canephora was obtained on Murashige and Skoog (MS) medium supplemented with 40 μM silver nitrate (AgNO3) and growth regulators indole-3-acetic acid (IAA) and N6 benzyladenine (BA). The highest response to shoot differentiation of 60% frequency and the maximum number of multiple shoots (2–3) per explant were obtained on MS medium containing 8.87 μM BA and 2.85 μM IAA. Apart from this, 70% of hypocotyl explants produced yellow friable embryogenic callus and also globular primary somatic embryos. Subsequent transfer onto the same medium induced secondary somatic embryogenesis. The micro-shoots, upon transfer to the same medium, in the following 6 weeks developed into 4-cm-long shoots with a single root. Further subculturing onto the same medium induced 4–5 roots in a 4-week period. The resulting plantlets were hardened and transferred to micro-pots containing sand:compost mixture (1:2), where 65% of them survived and resumed growth. By using optimal levels of AgNO3, it was possible to obtain effective direct organogenesis and embryogenesis. This system was used for genetic transformation using Agrobacterium tumefaciens. A stable transformation frequency of 2–5% was obtained when both types of explants, i.e., hypocotyl explants with collar region or hypocotyl explants without collar region, were co-cultivated with A. tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector. This is the first report of a hypocotyl collar region-based Agrobacterium-mediated transformation protocol for the economically important tropical plant C. canephora.

Rapid in vitro multiplication of Melia azedarach L. (a multipurpose woody tree)

An efficient regeneration protocol for rapid multiplication of Melia azedarach, an economically as well as medicinally important timber-yielding tree, was developed. Nearly 90% of the culture exhibited axillary bud sprouting and multiple shoot formation from nodal segments derived from 20-year-old candidate plus tree on Murashige and Skoog (MS) medium supplemented with 5 μM 6-benzyladenine (BA). The highest shoot regeneration frequency (92%), maximum number of multiple shoots (19.7 ± 0.31) as well as shoot length (4.9 ± 0.08 cm) was induced from nodal explants on MS medium amended with 5.0 μM BA, 0.5 μM indole-3-acetic acid (IAA) and 30 μM adenine sulfate (AdS). Addition of 250 mg l−1 ammonium sulphate, (NH4)2SO4, and 100 mg l−1 K2SO4, prevented defoliation and tip burning without affecting the number of shoots. The explant harvest period also influenced the bud break and shoot sprouting from nodal segments. Repeated subculturing of nodal explants on fresh MS medium containing lower concentration of BA (2.5 μM) along with IAA (0.5 μM), AdS (30 μM) and additives was found most suitable growth regulator regime for achieving 1.2-fold increase in shoot multiplication rate. The percentage of shoot multiplication as well as the number of shoots per node remained the same during first three subculture passages, afterwards a decline was recorded. About 90% of the in vitro regenerated shoots were successfully rooted ex vitro by giving a pulse treatment of 250 μM indole-3-butyric acid for 15 min, followed by their transfer to thermocol cups containing soilrite. The raised plantlets were successfully acclimatized first under culture room conditions, then to green house with 85% survival rate.

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

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

Plantlet production from the male inflorescence tips of Musa acuminata cultivars from South India

Inflorescence apices are suitable explants for the rapid in vitro propagation of Musa spp. However, the diploid and triploid banana cultivars showed different in vitro responses with respect to the hormone combinations in Murashige and Skoog medium. The diploid cultivar (Sannachenkadali, AA) induced a maximum number of multiple shoots in 8.9 μM 6-benzyl adenine (BA) whereas the triploid cultivar (Red banana, AAA) exhibited maximum multiplication in 22.2 μM 6-benzyl adenine. MS medium supplemented with 11.4 μM indole acetic acid and 17.8 μM BA was also suitable for shoot proliferation in triploid cultivar but not in the diploid cultivar. The regenerated shoots were rooted in Murashige and Skoog basal medium within 10–15 days. The rooted plantlets were transferred to vermiculite and maintained at a temperature of 25 ± 2°C for 10 days and then at room temperature (30–32°C) for 2 weeks before transferring to potted soil compost mixture. The plantlets showed 100% survival.

In vitro shoot multiplication through shoot tip cultures of Decalepis hamiltonii Wight & Arn., a threatened plant endemic to Southern India

An efficient and rapid micropropagation system was developed for a food and medicinally important endangered shrub, Decalepis hamiltonii (‘swallow root’), through shoot multiplication. The influence of 2.5–7.5 μM isopentenyladenine (2iP), 4.4–17.7 μM 6-benzyladenine, 2.3–4.7 μM kinetin, 2.8–6.8 μM thidiazuron, and 2.3–11.4 μM zeatin alone and in combination with 0.3–0.9 μM indole-3-acetic acid (IAA) on in vitro multiple shoot production was studied. The maximum number of multiple shoots (6.5±0.4) was induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.9 μM 2iP. But, both zeatin (9.1 μM) and kinetin (4.7 μM) in combination with IAA (0.6 μM) were able to produce a maximum of 5.0±0.4 and 5.1±0.4 multiple shoots, respectively. Further elongation of shoots and adventitious shoot formation was obtained on medium containing 2.5 μM 2iP and 0.3 μM gibberellic acid. Elongated shoots were separated and rooted on MS medium supplemented with 9.8μM indole-3-butyric acid (IBA) and various phenolic compounds within 5–6 wk. Phloroglucinol and salicylic acid interaction with IBA stimulated in vitro rooting of shoots. Successful field transfer was achieved in rooted plantlets.

Multiple Shoot Induction and Plant Regeneration from Embryo Axes of Six Cultivars of Gossypium hirsutum

The report describes in vitro plant regeneration from embryo axis explants of six cultivars of cotton. Induction of a maximum number of multiple shoots in all six cultivars could be achieved on Murashige and Skoog's (MS) salts and Gamborg's (B5) vitamins supplemented with 0.4 μM benzyladenine (BA) and 0.1 μM napthaleneacetic acid (NAA). Elongated shoots could be rooted on half strength medium supplemented with 0.5 μM NAA. Rooted shoots survived (92 %) after hardening in the greenhouse and grew to maturity (100 %) after transfer to field.

Micropropagation of Crossandra infundibuliformis (L.) Nees

Multiple shoots were induced from axillary and apical buds of Crossandra infundibuliformis (L.) on MS medium containing BAP and kinetin individually. The maximum number of multiple shoots were obtained from MS medium containing BAP 1 mg −1. The subcultures were done on fresh medium with the same composition and the number of multiple shoots increased during subculture of both types of explant. At the end of the second subculture, development of 45– 49 shoots per explant was noticed from the axillary bud culture, whereas apical buds produced 40–43 shoots per explant. Maximum elongation was obtained on the same medium but during subsequent subcultures. Elongated shoots were harvested and transferred to MS medium containing IBA 1.0 mg −1 for root induction. Rooted plantlets were transplanted to soil.

In Vitro Callus Culture and Plant Regeneration from Different Explants of Groundnut(Arachis hypogaea L.).

Callus induction and morphogenesis. from different groundnut explants were tested on MS salts supplemented with B5 vitamins, different concentrations and combinations of IAA, NAA. IBA, KIN and BAP. The explants were hypocotry, epicotyl, axillary bud, cotyiedonary node, immature leaf and immature embryos. The frequency of callus induction increased with increasing concentrations of auxins, the optimal levels being 3.0 mg/l of IAA or NAA and 0.5 mg/l of KlN. Among the explants, immature leaf was found to be most efficient explant in producing callus. Shoots were induced from callus cultures of hypocotyl, epicotyl, axillary bud, cotyledonary node, immature leaf and immature embryos with varying frequencies in the medium containing IAA or NAA (0.5 mg/l ) and KIN or BAP (0.5 - 2.0 mg/l ). Maximum number of multiple shoots were obtained from cotyledonary node explant. The regenerated shoots rooted best on MS medium containing 2.0 mg/l of IBA and 0.2 mg/l of KIN. Regenerated plants were norrrlially fertile. In vitro produced plantlets were transferred to soil and placed in glass house and they developed successfully, matured, and set seed. No phenotypic variants were observed among any of the plants produced in these experiments. Thus, this regeneration system offers an additional and more efficient tool for groundnut biotechnology research promrammes.

<i>In vitro</i> Regeneration in Three Varieties of White jute (<i>Corchorus capsularis</i> L.)

Healthy multiple shoot regeneration was observed from petiole-attached cotyledon (PC) explants of CVL-1 on MS containing 0.2 mg/l BAP and 1.0 mg/l IAA. On the other hand, the best response for multiple shoot regeneration in CVE-3 was obtained when the same explants were cultured on MS with 2.5 mg/l BAP and 0.5 mg/l NAA. However, the same explant of D-154 was found to show less responsive compared to other two varieties to produce multiple shoots. Cotyledonary nodal explants of all the three varieties were found to produce maximum number of multiple shoots on MS supplemented with 0.2 mg/l BAP and 1.0 mg/l IAA. Best root induction was observed at the base of the in vitro regenerated shoots on half the strength of MS supplemented with 0.3 mg/l IBA. The in vitro grown plantlets were successfully transplanted into soil. They grew up to maturity, flowered and fruited like the control plants.Key words: Regeneration, White jute, Corchorus capsularisDOI = 10.3329/ptcb.v17i1.1116Plant Tissue Cult. & Biotech. 17(1): 11-18, 2007 (June)

Effect of Bavistin and Adenine Sulphate on In vitro Shoot Multiplication of Picrorhiza scrophulariiflora

An alternative protocol for in vitro propagation of Picrorhiza scrophulariiflora is described using bavistin and adenine sulphate. The explants differentiated into multiple shoot buds on MS supplemented with various concentrations of bavistin and adenine sulphate ranging from 0 - 400 mg/l either alone or in combination. Maximum number of multiple shoots were obtained on MS containing the combination of bavistin (100 mg/l) and adenine sulphate (100 mg/l). In this combination as high as 28 shoots per explant was achieved and also vetrification of the cultures were not recorded. This study also demonstrates that the bavistin has stronger cytokinin-like activity than adenine sulphate. For instance, it was observed that bavistin alone in the concentration of 300 mg/l produced as high as 24 shoots per explant, however, adenine sulphate (100 mg/l) could produce a maximum of 18 shoots per explant. Moreover, higher or lower concentration did not improve the shoot multiplication. The microshoots were separated from the multiple shoots and transferred to MS containing various concentrations of auxins. Among them, NAA (1 mg/l) produced as high as 6 roots per explant. The regenerated plantlets were hardened in plastic cups (6 x 8 cm) containing 9 : 1 virgin soil and soil at Kyongnosla nursery and acclimated for four weeks. A 90% survival rate of the plants was recorded after 60 days. D.O.I. 10.3329/ptcb.v19i2.5441 Plant Tissue Cult. & Biotech. 19(2): 237-245, 2009 (December)