An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

Summary An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA₃) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.     

Regeneration and micrografting of lentil shoots

Summary A protocol for regeneration and micrografting of shoots of lentil (Lens culinaris Medik) was developed. Multiple shoots (4–5) were regenerated from cotyledonary node explants on Murashige and Skoog (MS) medium containing 8.8 μM 6-benzylaminopurine. In vitro regenerated shoots were micrografted on rootstocks with 96% efficiency. The successful grafts were transplanted to pots in Redi-earth^TM, hardened off and were grown to maturity with 100% success. The success of the micrografting was independent of the nature and concentration of growth regulator used in shoot initiation medium and the time period for induction of shoots. The protocol was successful with several cultivars of lentil. The advantages of micrografting over in vitro rooting are discussed.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

Thidiazuron-induced high-frequency axillary and adventitious shoot regeneration in Vigna radiata (L.) Wilczek

Summary Prolific shoot regeneration was achieved in mungbean Vigna radiata (L.) Wilczek from 3-d-old in vitro cotyledonary node and hypocotyl explants from seedlings derived from mature seeds on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.9 μM). An initial exposure to TDZ for 20 d and three successive transfers to fresh medium with reduced thidiazuron levels (0.09 μM) resulted in the regeneration of 104 shoots/explant from the cotyledon and 30 shoots/explant from the hypocotyl. Thidiazuron-associated abnormalities such as short compact shoots, fasciation and leaf growth in the form of rosettes were observed in shoots regenerated from hypocotyl explants. Both axillary and adventitious shoot formation from the explants were confirmed by histology. Through repectitive cycles of regeneration in the presence of TDZ, the number of shoots that could be obtained from the two explant classes within 80 d was significantly higher than with previous reports in mungbean     

Responsive regions for direct organogenesis in sunflower cotyledons

Regeneration efficiency from three different regions of cotyledonary explants was examined in six sunflower inbred lines. Proximal, middle and distal regions from seedling cotyledons were cultured on regeneration medium supplemented with growth regulators. Plant regeneration by direct organogenesis was observed after four weeks. Significant differences among inbred lines were found for regeneration percentage and average number of shoots per total explants. Also a decreasing regeneration capacity was observed from proximal to distal sections for all inbred lines. Regeneration ability from cotyledonary explants in this species is strongly influenced by the genotype and by the region from which the explant was obtained. The distance to the cotyledonary node plays a preponderant role in the expression of shoot forming capacity. Shoot differentiation via seedling cotyledons depends upon the presence of the proximal region of cotyledon regardless of the genotype.     

Rapid and efficient regeneration in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill] from whole cotyledonary node explants

A rapid and efficient regeneration protocol was established for soybeans [Glycine max (L.) Merrill]. Whole cotyledonary node explants were collected from aseptic seedlings cultured on MSB₅ medium containing 0.4 mg l⁻¹ N⁶-benzyladenine (BA). The effects of the plant growth regulators BA, kinetin (KT), indole-3-butyric acid (IBA) as well as the explant genotype on shoot regeneration were evaluated by the orthogonal design [L₁₆(4⁵)]. The process of shoot development was also observed. The regenerated shoots were elongated on MSB₅ medium and sufficiently elongated shoots were rooted on MSB₅ medium containing 0.5 mg l⁻¹ IBA. The results showed that all three of the plant growth regulators significantly affected shoot regeneration, with BA exhibiting the greatest benefit. The best combination for shoot regeneration was MSB₅ medium supplemented with 3.0 mg l⁻¹ BA , 0.2 mg l⁻¹ IBA and 0.5 mg l⁻¹ KT on Hefeng 25 genotype. Under these most favorable conditions, one explant could regenerate 30–35 shoots. Plantlets could be obtained within 2 months. The result of histocytological analysis indicated that protein accumulated gradually and reached to peak at late shoot bud formation.     

Overexpression of <Emphasis Type="Italic">AtATM3</Emphasis> in <Emphasis Type="Italic">Brassica juncea</Emphasis> confers enhanced heavy metal tolerance and accumulation

The eucalypt Corymbia torelliana × C. citriodora is planted widely in India, Brazil and Australia although plantation establishment has been limited by inadequate seed supply and low amenability to propagation via cuttings. This study optimised node culture and organogenic culture methods for in vitro propagation of Corymbia hybrids by identifying explant position (topophysic) effects on rooting, shoot elongation and shoot proliferation. Strong, negative morphogenic gradients in shoot elongation and proliferation capacity were evident from the cotyledonary node to the fourth or fifth node of seedlings when their nodes were transferred to node culture (without benzyladenine). These topophysic effects were related to differences in rooting capacity of individual nodes. Root formation in node culture was associated with formation of long multi-nodal axillary shoots, and so higher rooting of shoots from the cotyledonary node or first true-leaf node was associated with higher shoot proliferation. However, all nodes were equally capable of shoot proliferation in organogenic culture (with 2.2 μM benzyladenine), where rooting and rapid stem elongation did not occur. Most shoots (61–100%) from both node culture and organogenic culture were converted to plantlets, with plantlet conversion and primary root number not differing significantly among explant node positions. The strong topophysic effect in node culture, combined with the lack of a topophysic effect in organogenic culture, provides for an optimised clonal propagation system based on segregation of nodes from the same seedling into separate node and organogenic culture pathways.     

Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Whole plant regeneration from the shoot apex ofArachis hypogaea L.

Summary Arachis hypogaea L. peanut, has been a difficult species to manipulate in tissue culture. Lack of a reliable and quick regeneration method for peanuts has proven to be one of the hindrances in the application of transformation protocols to the crop. A protocol to initiate shoot apex elongation and rooting of these shoots is described. This protocol was successful with two peanut cultivars. Shoot apices were isolated from germinated seedlings and placed on Murashige and Skoog salts containing N⁶-benzyladenine for shoot initiation. Once shoot elongation occurred, the explant was transferred to a rooting medium containing Murashige and Skoog salts and only one plant growth regulator, α-naphthalene acetic acid. In as few as 3 weeks, the explants began to root and could be transferred to soil. Forty-five percent of explants isolated from germinating peanut seeds would root on this medium. Elongation and rooting of the shoot apices were not hindered by the addition of an antibiotic to the medium, indicating that the regeneration method could be useful inAgrobacterium tume-faciens-mediated transformation protocols.     

Clonal propagation of mesquite tree (<Emphasis Type="Italic">Prosopis laevigata</Emphasis> Humb. &; Bonpl. ex Willd. M.C. Johnston). I. Via cotyledonary nodes

Plantlet regeneration in Prosopis laevigata (Humb. & Bonpl. ex Willd.) Johnston (Fabaceae), a multipurpose tree, has been achieved from cotyledonary nodes excised from in vitro grown seedlings. The explants were cultured on MS media containing different concentrations of N-6 benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-d) and a mixture of organic components. The highest number (3.37 + 0.51) of multiple shoots was observed in MS media containing 2,4-d (9.05 μM) + BA (6.62 μM). The regenerated shoots were then transferred onto half-strength MS medium containing a plant growth regulator that was either: indole-3-butyric acid, 1-naphthaleneacetic, indole-3-acetic acid, or 2,4-d as well as phytagel or vermiculite for adventitious root initiation. Best rooting efficiency of 44.0% was obtained when NAA (16.11 μM) and vermiculite were used. After rooting, the cloned plantlets were successfully hardened to ex vitro conditions. This work may help to reduce the devastation caused by the overexploitation of this species.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

Differences in shoot regeneration response from cotyledonary node explants in Asiatic Vigna species support genomic grouping within subgenus Ceratotropis (Piper) Verdc.

The efficiency of any plant regeneration system lies in part in its wide applicability to diverse genotypes. In Asiatic Vigna, cotyledon and cotyledonary node explants from 4-day-old seedlings of 27 genotypes were cultured in a medium consisting of MS salts, B5 vitamins, 3.0% sucrose and 1.0 mg l^-1 BA. Direct and efficient multiple shoot regeneration (80–100%) from the cotyledonary nodes was obtained in all epigeal species namely radiata, mungo, aconitifolia, subspecies radiata var. sublobata, mungo var. silvestris and in the hypogeal but allotetraploid glabrescens. In contrast, two other hypogeal species V. angularis and V. umbellata failed to initiate shoots from the nodes. However, adventititious shoots developed at the basipetal cut (hypocotyl) in 35–67% of V. angularis explants. These results provide evidence in support of the existing genomic grouping within subgenus Ceratotropis, which designates AA, A₁A₁ and A₁A₁/- to epigeal, hypogeal and the allotetraploid species, respectively. Mean shoot production ranged from 3.3 to 10.4 shoots per explant during the first subculture and varied significantly among the responsive genotypes within 4 species. Additional shoots were obtained in all genotypes after subsequent subculture. However, cotyledons were not as regenerable as cotyledonary node explants. Although significant differences in rooting were observed among the shoots of the 15 genotypes, the response was generally higher in MS basal medium (MSO) than in MS with 1.0 mg l^-1 IAA. Regenerated plants were successfully transferred to soil (50–100% survival rate) and all surviving plants were reproductively fertile. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of Albizia odoratissima L.F. (Benth.) from cotyledonary node and leaf nodal explants

Summary Cotyledonary node and leaf nodal explants excised from 14-d-old in vitro-grown seedlings of Albizia odoratissima were cultured on a Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP), N ⁶-(2-isopentenyl) adenine (2-iP) and kinetin, used either solely or in combinations. The highest frequency for shoot regeneration (82.5%), the maximum number of shoots per explant (6.9), and the maximum shoot length (2.55 cm) were obtained from cotyledonary node explants cultured on a MS medium containing 10 &#x03BC;M BAP and 10 &#x03BC;M 2-iP with 30 gl^&#x2212;1 sucrose. Successful rooting was achieved by placing the microshoots on MS medium with 25 &#x03BC;M indole-3-butyric acid (IBA) for 24h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was best for plant acclimatization, in which 75% of plants survived.     

An efficient plant regeneration system for Mucuna pruriens L. (DC.) using cotyledonary node explants

Summary The purpose of this study was to develop an efficient micropropagation system for Mucuna pruriens, an important medicinal plant in India. A range of cytokinins was investigated for multiple shoot regeneration with cotyledonary node explants from 7-d-old aseptic seedlings. Of all the cytokinins, 6-benzyladenine (BA), kinetin (KIN) and 2-isopentenyl adenine (2-iP) tested in Murashige and Skoog medium (MS), BA was the most effective and 5.0 μM was found to be optimum for inducing maximum shoots. Medium types, medium strength and pH were also investigated for induction and proliferation of shoots. The highest efficiency of shoot proliferation was observed in 5.0 μM BA and 0.5 μM α-naphthalene acetic acid (NAA) in half-strength MS medium at pH 5.8. The best condition for rooting was half-strength MS medium solidified with agar and with 2.0 μM indole-3-butyric acid (IBA). After rooting, the plantlets were transferred to plastic pots filled with sterile soilrite where 90% grew and all exhibited normal development.     

Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes

A protocol is presented for micropropagation of an economically important timber-yielding forest tree, Dalbergia sissoo Roxb. (Sissoo). Multiple shoots were induced from cotyledonary nodes derived from 1-week-old axenic seedlings on Murashige and Skoog's medium containing either N ⁶-benzyladenine (BA), kinetin (Kn), isopentenyladenine (2iP) or thidiazuron (TDZ), with BA being the most effective growth regulator. High-frequency shoot proliferation (99%) and maximum number of shoots per explant (7.9 shoots) were recorded with BA at an optimum level of 8.9 μM. Concentrations of all cytokinins tested above the optimum level markedly reduced the frequency of shoot proliferation. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary node on shoot multiplication medium after each harvest of the newly formed shoots. Primary shoots were multiplied as nodal explants, and from each stem node 2 or 3 shoots developed. Thus, 60–70 shoots were obtained in 3 months from a single cotyledonary node. About 91% of the shoots developed roots following transfer to half-strength MS medium containing a combination of 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. Eighty percent of the plantlets were successfully acclimatized and established in soil. Received: 1 October 1997 / Revision received: 31 March 1998 / Accepted: 7 April 1998     

Micropropagation of Capparis decidua through In Vitro Shoot Proliferation on Nodal Explants of Mature Tree and Seedling Explants

In vitro clonal propagation of Capparis decidua was achieved using nodal explants from mature trees, and cotyledonary node, cotyledon and hypocotyl explants taken from the seedlings. Explants cultured on MS medium supplemented with BAP showed differentiation of multiple shoots and shoot buds in 4–5 weeks in the primary cultures. The medium with BAP (5 mg/l) was the best for shoot bud proliferation from the nodal as well as seedling explant. Shoot multiplication was best on cotyledonary node. In the nodal explants shoot multiplication was best on medium supplemented with 5 mg/l BAP and after second subculturing further multiplication of shoot buds was highest on the medium containing 3 mg/l BAP. Shoots were separated from mother cultures in each subculturing for rooting. Rooting was best achieved using 1 mg/l IBA in the medium. Rooted plantlets were transferred td earthen pots with garden soil and peat moss mixture.     

Micropropagation of <Emphasis Type="Italic">Pongamia pinnata</Emphasis> through enhanced axillary branching

A complete protocol is presented for the first time for the micropropagation of Pongamia pinnata, a biofuel tree, using cotyledonary nodes derived from axenic seedlings. Multiple shoots were induced in vitro from nodal segments through forced axillary branching. Murashige and Skoog (MS) medium supplemented with 7.5 μM benzylaminopurine (BAP) induced up to 6.8 shoots per node with an average shoot length of 0.67 cm in 12 d. Incorporation of 2.5 μM gibberellic acid (GA₃) in the medium during the first subculture after establishment and initiation of shoot buds significantly improved the shoot elongation. Single use of GA₃ during the first subculture eliminated the need for prolonged culturing on BAP medium. Further use of GA₃ in the medium was not useful. Shoot culture was established for at least two subcultures without loss of vigor by repeatedly subculturing the original cotyledonary node on shoot multiplication medium followed by shoot elongation medium after each harvest of the newly formed shoots. Thus, from a single cotyledonary node, about 16–18 shoots were obtained in 60 d. Shoots formed in vitro were rooted on full-strength MS medium supplemented with 1.0 μM indole butyric acid (IBA). Plantlets were successfully acclimated, established in soil, and transferred to the nursery.     

High-frequency multiple shoot regeneration from cotyledonary nodes of guar (Cyamopsis tetragonoloba L. Taub)

Summary Guar (Cyamopsis tetragonoloba L. Taub) is a drought-tolerant multipurpose cash crop. A rapid regeneration system has been developed for four Indian guar genotypes. Investigations were carried out to assess the effect of different growth regulators and their combinations on a variety of explants such as the embryo, cotyledons, and cotyledonary nodes for shoot morphogenesis. It was established that Murashige and Skoog's culture medium containing 6-benzylaminopurine (13.3 μM or 3 mgl⁻¹) in combination with indole-3-acetic acid (11.4 μM or 2mgl⁻¹) with cotyledonary node explants gave the highest frequency of multiple shoot induction. In vitro rooting from cultured shoots was maximal on a half-salt concentration of Murashige and Skoog's culture medium fortified with indole-3-butyric acid (4.9 μM or 1 mgl⁻¹). In vitro-regenerated plants were grown to pod setting and subsequent maturity in greenhouse conditions.     

Establishment of efficient and rapid regeneration system for some diploid wild species of <Emphasis Type="Italic">Arachis</Emphasis>

Though peanut tissue culture has advanced to a considerable extent using a number of explants with the subsequent production of transgenic plants, wild Arachis species appeared to be recalcitrant to using similar explants. In this study, the use of cotyledonary nodes as explants prepared from 7-day old seedlings resulted in the development of a simple and rapid regeneration protocol for five diploid wild species including A. diogoi, A. stenosperma, A. duranensis, A. cardenasii and A. correntina belonging to the genus Arachis for producing multiple shoots. Shoot bud initiation was observed 10 to 15 days after culture initiation. Responding cotyledonary nodes with shoot buds were subcultured to lower levels of cytokinin and finally to MS basal medium for further shoot development and elongation. Production of multiple shoots was observed in all the five diploid species with a maximum of 9 to 16 shoots were obtained per explant in the primary cultures. The number of shoot buds increased significantly with repeated explant subculturing with recovery up to 45 shoots from responding explants. These shoots were rooted efficiently on MS medium supplemented with 1 mg l⁻¹ naphthalene acetic acid and the time taken from explanting to the transfer of shoots to potting mixture was about 12 weeks. All rooted shoots were successfully established in soil in glass house and further transferred to field. These plants survived to maturity and set seed.     

Thidiazuron promotes in vitro regeneration of wheat and barley

Summary Thidiazuron (TDZ) is a substituted phenylurea which has been shown to be an efficacious regulator of in vitro morphogenesis of many dicot plant species. However, information regarding the effect of TDZ on in vitro regeneration of monocot species is limited. We investigated the effects of TDZ on in vitro regeneration of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) and found that it promoted shoot regeneration from callus in these two important cereal species. Plant regeneration from calluses derived from immature embryo culture of barley and wheat was observed in regeneration media with a wide range of TDZ concentrations (0.045–45 μM). Shoot regeneration from barley calluses was the highest (38.3% for cv. Golden Promise) at 4.5 μM (1 mg l⁻¹) TDZ, while the optimal TDZ level for wheat regeneration seemed to be 0.9 μM (0.2 mg l⁻¹) (87% for cv. Bob White and 49.4% for cv. Hi Line). Roots developed normally when the regenerated wheat and barley shoots from TDZ-containing media were transferred to the rooting medium. Comparison with other plant growth regulators commonly used in wheat and barley regeneration media suggested that TDZ was among the best for in vitro regeneration of wheat and barley. Both authors contributed equally