Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

High frequency plantlets regeneration from seedling explants of Prosopis tamarugo

Callus cultures of Prosopis tamarugo Phil (Leguminosae, Sub family-Mimosoideae) were established from hypocotyls and cotyledons on MS medium supplemented with NAA (2.0 mg l^-1) and BAP (0.2 mg l^-1). Regeneration through various juvenile explants was obtained on hormone-free and high cytokinin containing Murashige and Skoog's medium. Multiple shoot buds formation was observed from the embryonic axis on MS medium incorporated with BAP (5.0 mg l^-1)). Elongation of shoot buds was observed on subsequent transfer to MS medium with BAP (1.0–2.5 mg l^-1) or without BAP. Explants containing apical meristem showed higher number of shoot formation at an early period. De novo shoot buds formation through callus morphogenesis was observed at the base of differentiated shoots on high cytokinin containing medium. All the manipulations of salt strength of MS, nitrogen, carbon, ascorbic acid and polyamines failed to induce organogenesis in isolated callus. In vitro produced shoots were rooted on MS medium supplemented with IBA or NAA singly or in combination.Abbreviations HC high cytokinin (BAP 5.0 mg l^-1) - BAP 6-benzyl amino purine - IBA indole-3-butyric acid - HF hormone free - NAA I-naphthalene acetic acid - MS Murashige & Skoog     

Direct shoot organogenesis and plant regeneration in safflower

Summary  Adventitious shoot buds were induced directly on the adaxial surface of the cotyledons of eight safflower cultivars after 14 d of culture initiation on Murashige and Skoog's (MS) medium supplemented with various levels of 6-benzylaminopurine (BA). Maximum shoot organogenesis of 54.4% with 10.2 shoots per responding cotyledon was obtained with 8.87 μM BA in the cv. S-144. Regenerated shoots were classified into three groups on the basis of their morphological features and were found to be correlated with the levels of BA. The highest number of normal shoots was obtained from 2.2 μM BA treatment. The regenerated shoots of group I (normal shoots) were rooted on half-strength MS medium supplemented with 5.3 μM α-naphthaleneacetic acid, 3% sucrose and 0.8% bacto-agar. Rooted plantlets were successfully transferred to soil and appeared morphologically normal. Histological studies revealed that shoot buds originated adventitiously from subepidermal cells.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct shoot bud induction and plant regeneration in <Emphasis Type="Italic">Capsicum frutescens</Emphasis> Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 μM indole-3-acetic acid, 10 μM silver nitrate and either of 13.31–89.77 μM benzyl adenine (BA), 9.29–23.23 μM kinetin, 0.91–9.12 μM zeatin, 2.46–9.84 μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 μM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.     

In vitro clonal multiplication of mature trees of Dalbergia sissoo Roxb.

Multiple shoots were obtained from nodal explants of 30-year-old trees of Dalbergia sissoo Roxb. on a defined medium, MS (Murashige & Skoog medium) supplemented with auxin-cytokinin combinations. IAA (Indole accetic acid) alone promoted 15% rooted shoot buds. A combination of IAA+Kn (Kinetin) gave 100% rooted shoot buds. A combination of NAA (napthaleneacetic acid) + Kn and NAA+BAP (6-benzylaminopurine) also gave high percentages of rooted shoot buds. Ascorbic acid in the medium prevented the death of callus and plantlets, which followed darkening of the medium.     

Gradual Depletion of 2,4-D in the Culture Medium for Indirect Shoot Regeneration from Leaf Explants of Camellia sinensis (L.) O. Kuntze

Adventitious shoot regeneration via callus phase from in vitro leaf explants is reported for the first time in tea. Callus was obtained on Murashige and Skoog medium supplemented with varied concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5, 5.0, 7.5 and 10.0 mg/l). Rhizogenesis was observed at all concentrations of 2,4-D. Adventitious shoot buds developed indirectly on leaf explants after prolonged culture for 16 weeks on medium supplemented with 10.0 mg/l 2,4-D. GC analysis of the medium and the tissues at different stages of development showed that specific levels of 2,4-D in the tissue were responsible for morphogenesis. Shoot buds developed on rhizogenic calli, only when 2,4-D declined to undetectable or negligible concentrations in the tissue probably due to detoxification and metabolism. Alternatively, shoot buds could also be evoked when rhizogenic calli were transferred to medium supplemented with low concentration of 2,4-D (1.5 mg/l). The adventitious nature of the shoots was confirmed through histological studies.     

Tissue culture and plant regeneration of okra (Abelmoshus esculentus)

Explants (hypocotyl, cotyledon, cotyledonary node and leaf segment) were excised from aseptically grown okra (Abelmoschus esculentus) seedlings. The explants were cultured on a Murashige and Skoog basal nutrient medium supplemented with auxins, cytokinins and auxin-cytokinin combinations. Callus formation and root differentiation occurred in a medium containing naphthaleneacetic acid (NAA) or indoleacetic acid. There was a greater proliferation of roots on medium supplemented with NAA. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to the growth medium suppressed root formation. No shoot bud or shoot development was observed at any of the auxin levels tested. Both kinetin (KN) and zeatin (Z) also proved ineffective in inducing shoot buds or shoots. Shoots were produced on cotyledon and cotyledonary node explants cultured in a medium supplemented with benzyladenine and NAA. These shoots developed roots on the same medium. The plantlets, on transfer to soil, grew normally.     

Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

Plant regeneration from different explants of neem

When different seedling explants, i.e. hypocotyl, epicotyl, cotyledonary node, root-shoot zone, cotyledon, leaves and roots from 7-day-old seedlings of neem were cultured on Murashige and Skoog's medium supplemented with 2 mg l⁻¹ benzyladenine and 0.1 mg l⁻¹indole-3-acetic acid, shoot buds were initiated from all the explants tested, with leaf explants producing the highest average number of shoots/explant. The regenerated shoots were further subcultured and later could be rooted on a medium supplemented with indole butyric acid (1 mg l⁻¹) and complete plants could be obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

High copper levels in the medium improves shoot bud differentiation and elongation from the cultured cotyledons of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L.

The effect of copper sulphate on differentiation and elongation of shoot buds from cotyledonary explants of Capsicum annuum L. cv X-235 was investigated. Shoot buds were induced on medium supplemented with 22.2 μM BAP and 14.7 μM PAA. Elongation of shoot buds was obtained on MS medium containing 13.3 μM BAP + 0.58 μM GA₃. Both shoot induction and elongation media were supplemented with different levels of CuSO₄ (0–5 μM). The levels of CuSO₄ in the induction as well as elongation medium highly influenced the shoot bud formation and their subsequent elongation. Highest number of shoot buds per explant was obtained when the concentration of CuSO₄ was increased 30 times to the normal MS level. Shoot buds formation frequency i.e., the number of shoots formed per explant was increased two fold as compared to those formed on control. Elongation both in terms of percentage and length of shoots was better than that on control. Healthy elongated shoots were rooted on MS medium supplemented with 5.7 μM IAA. Rooted plantlets were transferred to field conditions.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

In vitro differentiation of multiple shoot clumps from intact seedlings of switchgrass

Summary An efficient and reproduciblein vitro culture system has been developed for regeneration of multiple shoot clumps from intact seedlings of both lowland and upland cultivars of switchgrass (Panicum virgatum L.). The multiple shoots were induced on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-phenyl-3-(1,2,3-thiadiazol-5YL)-urea (thidiazuron or TDZ). Maximum response was obtained with 4.5 μM 2,4-D and 18.2 μM TDZ. These shoots proliferated and rooted efficiently on MS medium without growth regulators. The developmental pattern of the multiple shoots indicated their origin from the enlarged shoot apex via proliferation of axillary buds and subsequent reprogramming of shoot meristems followed by secondary differentiation of adventitious shoots The simplicity of the protocol and direct production of multiple shoots make this a potential system that is highly attractive and amenable for microprojectile-mediated gene transfer.     

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     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.