Precocious gladiolus corm formation in liquid shake cultures

Conditions were defined for precocious differentiation and improved growth of corms at the base of gladiolus shoots. Shoots were derived from explants cultured on agar solidified media, and corm regeneration was obtained in subsequent liquid shake cultures. Benzyladenine (BA), at 10^-7 M, was found to have a stimulating effect mainly when provided to the shoots prior to manifestation of corm growth. Paclobutrazol and sucrose promoted corm formation when supplemented to the liquid media. Paclobutrazol, at 10 mg l^-1, shifted assimilate allocation towards the growing corm. A differential promotion of corm development by sucrose was not observed, and the concentration of sucrose at which the sugar demand for maximal shoot and corm growth is satisfied (60 g l^-1) was unaltered by the presence of paclobutrazol. The rate of corm growth on shoots cultured in a liquid medium supplemented with paclobutrazol and a saturating sucrose concentration, was a function of the length of the shoot's leaf blades, and was similar in light and in dark.     

In vitro cormlet development in Crocus sativus

An improved protocol for generation of viable cormlets from tissue culture derived shoots of saffron has been developed. Multiple shoots were generated from apical buds, small corms and in vitro developed single shoots. Bunches of two to three shoots when cultured on half strength Murashige and Skoog (MS) medium containing 3 mg dm⁻³ benzyladenine (BA) and 80 g dm⁻³ sucrose developed 1.89 cormlets per shoot bunch with an average fresh mass of 1.18 g. It took nine months from culture of apical buds to the harvest of cormlets but under field conditions 22 months. Sucrose appeared to be essential for cormlet induction as no cormlets were developed in the medium devoid of sucrose and only 0.29 per shoot in medium containing mannitol. In vitro derived cormlets sprouted from apical and axillary buds on MS medium containing 12 mg dm⁻³ BA, 3 mg dm⁻³ indolebutyric acid and 30 g dm⁻³ sucrose. Daughter cormlet formation from in vitro derived cormlets was also observed.     

Axillary shoot proliferation and in vitro flowering in an adult giant bamboo, Dendrocalamus giganteus Wall. Ex Munro

Summary Continuous axillary shoot proliferation and in vitro flowering were achieved using single node explants from a mature (over 70-yr-old) field clump of Dendrocalamus giganteus (giant bamboo). The shoots proliferated in a basal Murashige and Skoog medium with 6 mgl⁻¹ (26.6 μM) N⁶-benzyladenine (BA) and 2% sucrose. The rate of shoot proliferation gradually increased to over three-fold before in vitro flowering took place. In vitro flowering was not the expression of a species-specific mechanism believed to occur during gregarious flowering, as the mother clump did not flower. The rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation leading to build up, and release of stress in relation to flowering and its reversion are discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

In vitro regeneration of Alstroemeria cv. ‘Yellow King’ by direct organogenesis

The common techniques for the in vitro production of Alstroemeria plants are based on rhizomes as explants, which have low multiplication rates and a high risk of carrying viral diseases. To overcome these problems, we developed a protocol for the in vitro regeneration of Alstroemeria cv.‘Yellow King’, by testing for shoot induction several explant sources (leaf, stem apices, rhizomes and immature inflorescence apices), temperature and light/dark regimes, hormone and salt concentrations. For shoot multiplication and rooting, several hormone concentrations were tested. We found that only the young floral apices produced adventitious shoots by direct organogenesis. The highest shoot induction rate (10.4 shoots per explant) was obtained by incubation in the dark for 15 days at 8 °C followed by 15 days at 25 °C and a 16-h/8-h light/dark regime, on a Murashige and Skoog (1962) liquid medium at 50% of the salt concentration, supplemented with 2.5 mg l⁻¹ KIN, 1.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, using a piece filter paper to support the explant. The highest shoot multiplication rate (9 shoots per explant) was obtained on a liquid MS medium at full strength supplemented only with BA at 1.0 mg l⁻¹. In vitro rooting of shoots was induced also on a liquid MS medium, either with or without plant hormones.     

The role of sucrose and different cytokinins in the in vitro floral morphogenesis of rose (hybrid tea) cv. “First Prize”

Shoots of rose (hybrid tea) cv. “First Prize” were induced to flower in vitro on Murashige and Skoog (MS) medium containing various sucrose concentrations (15, 30 or 45 g l⁻¹) and different phytohormone combinations of different cytokinins [N⁶-benzyladenine (BA); thidiazuron (TDZ) and zeatin] with α-naphthaleneacetic acid (NAA). Results indicate that sucrose is the key factor in floral morphogenesis while cytokinin increases the flowering percentage and helps the normal development of floral buds. From the three cytokinins that were used, BA and zeatin were considered to be more suitable as inductive flowering agents than TDZ. Reduced inorganic and organic salt concentration in MS media had a positive effect on in vitro flowering. The morphology of shoots bearing floral buds varied with different cytokinin treatments. The highest percentage (45%) of flowering was obtained on MS medium supplemented with 3.0 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA and 30 g l⁻¹ sucrose.     

Globular embryo-like structures and highly efficient thidiazuron-induced multiple shoot formation in saffron (<Emphasis Type="Italic">Crocus sativus</Emphasis> L.)

Saffron (Crocus sativus L.) is a monocotyledonous plant propagated via corms, but recently several alternative methods have been reported. To find the conditions suitable for saffron shoot formation from corms, the effect of different concentrations of the plant growth regulatory cytokinins N⁶-benzyladenine (BA) and N-phenyl-1, 2,3-thidiazol-5-ylurea, commonly known as thidiazuron (TDZ), were compared. In all corm explants, an average of 39.5 ± 5.1 shoots per corm were induced by 4.54 μM TDZ, whereas only 3.6-11.4% by BA. The outstanding result in the shoot formation stage is the generation of globular, translucent structures that are morphologically similar to globular embryos. To optimize the plant regeneration from the induced adventitious shoots obtained from the TDZ treatment, the shoots were transferred to MS and B5 media supplemented with different concentrations and combinations of NAA and BA. The highest rate of plant regeneration from developing shoots was observed in the B5 medium containing 2.22 μM NAA and 2.68 μM BA. With optimized hormonal conditions, an average of 19.55 ± 5.75 shoots and 3.18 ± 1.5 roots per explants were obtained. Based on this experiment, a simple, new and efficient protocol is presented to produce numerous plants from induced corm explants of saffron.     

In vitro multiplication and microrhizome induction in Curcuma aromatica Salisb.

Shoot multiplication and plant regeneration was achieved from freshly sprouted shoots of Curcuma aromatica on Murashige and Skoog's medium supplemented with BA alone (1–7 mgL^–1) or a combination of BA(1–5 mgL^–1) and Kn (0.5–1 mgL^–1). A concentration of 5 mgL^–1 BA was optimum for shoot multiplication and rooting of shoots. The regenerated plants grew profusely on transfer to liquid medium.In vitro raised plants were successfully established in the field. Microrhizomes were induced at the base of the in vitro derived shoots upon transfer to medium containingvarious combinations and concentrations of sucrose and BA and grown under varying photoperiods. MS basal medium with 5 mgL^–1 BA, 60 gL^–1 sucrose and an8 h photoperiod was optimum for induction ofmicrorhizomes within 30 days of culture. Harvestedmicrorhizomes stored in moist sand in poly-bagssprouted after 2 months of storage at roomtemperature. For in vitro storage, microrhizomeswere grown in medium containing 0.1 mgL^–1 BA.Microrhizome formation was found to be controlled bythe concentrations of BA and sucrose as well asphotoperiod during culture.     

Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

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.     

An improved system for the <Emphasis Type="Italic">in vitro</Emphasis> regeneration of <Emphasis Type="Italic">Thapsia garganica</Emphasis> via direct organogenesis – influence of auxins and cytokinins

An improved protocol for mass multiplication directly from leaf material of Thapsia garganicawas developed. Using factorial experimentation, auxins (NAA, IAA, 2,4-D) and cytokinin (BA, kinetin) combinations at 0–2 mg l⁻¹ added to Murashige and Skoog (MS) medium with 30 g l⁻¹ sucrose and 8 g l⁻¹ agar (pH 5.8) were tested for their effect on direct regeneration on leaflet and petiole explants. Of the media tested, the 0.5:1.5 NAA:BA medium was comparable for direct shoot organogenesis to the 2 mg l⁻¹ kinetin supplemented medium. However, when shoots were multiplied on these media, the 2 mg l⁻¹ kinetin without auxins was most effective as it kept the percentage of callus-derived plantlets to 3% and the number of hyperhydric shoots were minimal at a frequency of 2% compared to 25% on the 0.5:1.5 NAA:BA medium. The 2 mg l⁻¹ kinetin medium induced adventitious bud formation in 36% of the explants after 30 days. When the cultures were transferred to the same medium for multiplication, an average of six shoots (4.3 cm) were derived from each shoot base. Other combinations resulted in callus formation that either preceded shoot production or occurred together with adventitious shoot induction; whereas the 2 mg l⁻¹ medium resulted solely in adventitious buds that readily converted and elongated to shoots. On the 0.5:1.5 NAA:BA medium which tended to induce hyperhydric shoots in culture, agar (0.8, [w/v]) (15% hyperhydric plantlets) was more useful in maintaining a high health status in regenerating plantlets than gellan gum (Gelrite^®; 0.25, [w/v]) (60% hyperhydric plantlets). Although rooting in vitro was difficult, 58% of the propagules were successfully acclimatized when plants were exposed to fungicidal solutions as pre- and post-acclimatization treatments. A comprehensive protocol that allows for a reduction in mortality due to damping-off diseases during ex vitro transplantation of the in vitro-derived T. garganica plantlets is reported. The acclimatization procedure presented here is potentially suited to other umbelliferous species where fungal rots hamper ex vitro establishment.     

In vitro propagation of cashew from young trees

Summary Regeneration of cashew (Anacardium occidentale L.) from shoot explants of young grafts of mature tree origin is described. Establishment of shoot cultures was affected by season of collection, source, and type of explant. Explants from young grafts established better than those collected from field trees, and nodal cuttings regenerated better than shoot tips. Maximum percentage bud break and minimum contamination was noticed when shoots were collected in dry months (January to May). Pre-conditioning of stock plants by hormonal spray with 6-benzyladenine (BA) and gibberellic acid (GA₃) and brief presoaking of shoots in BA had no significant effect on culture establishment. MS (Murashige and Skoog, 1962) medium with half-strength major nutrients, 2.74 mM l-glutamine, 87.6 mM sucrose, and 2.25 gl⁻¹ phytagel was ideal for culture initiation. Inclusion of 0.1% polyvinylpyrrolidone (PVP-360) in the media reduced phenolic exudation. Solidified media was superior to liquid medium. Sucrose/glucose as energy source was found essential in the medium and had significant effect on percentage bud break and shoot development. A repeatable axillary shoot-bud induction was obtained on the above basal medium containing thidiazuron (TDZ) alone and in combination with BA. TDZ at 0.45 μM was best for axillary shoot-bud proliferation (4.5 buds per shoot) with maximum response (100%). Bud elongation could be stimulated in multiple shoots on medium containing 116.8 mM sucrose. In vitro rooting on auxin media and pulsing microshoots in 10 mM naphthalenaacetic acid (NAA) was ineffective. Rooting inability was, however, overcome by a micrografting procedure.     

Factors influencing the growth of micropropagated shoots and in vitro flowering of gentian

About 70% of the shoots developed from nodal explants ofGentiana triflora flowered in vitroondouble strength WPM medium containing 3% (w/v) sucrose, 0.5mg/l BA after 12 weeks of culture in a growth room at 22°Cwith continuous illumination (PPFD=60molm^–2 s^–1). The influences oninvitro shoot development and flowering of several factors includingthe position of the explant, requirements for sucrose, cytokinin orGA₃, variations of pH and photosynthetic photon flux density (PPFD)were investigated. In vitro flowering but not shootdevelopment of G. triflora decreased notably withincreaseddistance from the apex of the shoot, indicating the presence of a floralgradient in the micropropagated shoots. Conversely, as little as 0.01mg l^–1 GA₃ in the medium promotedshootdevelopment but even up to 0.2 mg l^–1GA₃ did not induce in vitro flowering.Even though BA could substitute GA₃ for a high level of shootdevelopment, it also promoted a high level of in vitroflowering at the PPFD of 60 molm^–2 s^–1. Sucrose was required for shootdevelopment and flowering in vitro and higher levels ofPPFD could not compensate effectively for the omission of the sugar from themedium. In general, the effects of different concentrations of BA in the mediumor variations of pH on shoot development and flowering invitro were found to be influenced by PPFD. A novel observation isthat precocious flowering of micropropagated gentian shoots did not occur ifthey were first cultured for 5 weeks in the dark before transfer to the lightcondition.     

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.     

Micropropagation of adult Swainsona formosa (Leguminosae: Papilionoideae: Galegeae)

Summary Explants of axillary buds excised from mature adult stems of Swainsona formosa (G. Don) J. Thompson (syn. Clianthus formosus) were cultured on Murashige and Skoog medium supplemented with a range of auxins, cytokinins, and sucrose concentrations. Auxins did not increase shoot or bud numbers above controls, and 2,4-dichlorophenoxyacetic acid was the only auxin to significantly increase callus production. Benzyladenine or thidiazuron incorporated into the medium at 0.1 μM stimulated shoot and bud production, and shoot growth occurred following removal of cytokinins from the medium after 4 wk. Shoot number increased linearly with sucrose concentration up to 40 g l⁻¹, but shoot height and the number of cytokinin-induced buds were optimal at sucrose levels of 20–30 g l⁻¹. Roots were initiated in vitro following treatment of cuttings with 0.1% indole-3-butyric acid and 0.1% α-naphthaleneactic acid. Plantlets were successfully established in soil but were plagiotropic and exhibited distichous phyllotaxy.     

Regeneration of `juvenile' plants of black plum,Syzygium cuminii Skeels,from nodal exp of mature trees

Nodal explants, excised from young shoots from mature trees of Syzygium cuminii, were cultured on MS medium supplemented with different concentrations of BA or kinetin. Among these, BA (0.5 or 1.0 mg l^–1) induced greening and opening of the incipient shoot buds, which however did not elongate. Elongation of the shoot buds was facilitated on MS medium with 1.0 mg l^–1 BA supplemented with casein hydrolysate (1.5 g l^–1) or glutamine (200 mg l^–1). Nodal explants (microcuttings), taken from shoots developed in vitro, also developed multiple shoots when cultured on MS with1 mg l^–1 BA. These explants did not require an additional supply of reduced nitrogen, for further normal development. Shoots developed from explants from mature trees and microcuttings were rooted by sub-culturing them on Knop's medium supplemented with 2% sucrose and 1 mg l^–1 IAA. The plants that developed in vitro were planted in soil and were transferred to the field after an acclimatization period of 7–8 months. These plants have been thriving well for more than three years and have no apparent phenotypic aberrations.     

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

Summary 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.     

Shoot regeneration,re-flowering and post flowering survival in bamboo inflorescence culture

In vitro grown inflorescences of Bambusa edulis were used to investigate the process of vegetative shoot growth in detail. The findings revealed that auxins and ACC could be significant growth regulators in this process. Overall, auxins [NAA, indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)] induced inflorescences to grow vegetative shoots. However, the efficiency of shoot regeneration varied. A greater percentage (27.3–34.5) of inflorescences in the 5 mg l⁻¹ NAA, 10 mg l⁻¹ NAA, and 1 mg l⁻¹ 2,4-D treatments formed more vegetative shoots than those exposed to other treatments. IBA promoted shoot regeneration less effectively than NAA and 2,4-D. Fifty percent of regenerated vegetative shoots flowered after 2 months when the medium was supplemented with 5 mg l⁻¹ NAA. All shoots that received 1 mg l⁻¹ 1-amino-cyclopropane-1-carboxylic acid (ACC) flowered in 5 mg l⁻¹ NAA medium. Rooted plantlets were used to examine their survival following in vitro flowering. All plantlets with vegetative shoots, even those with inflorescences, survived and grew.     

In vitro micropropagation of a Cucurbita interspecific hybrid cultivar – a root stock plant

An efficient in vitro micropropagation protocol was developed for direct shoot growth of interspecific Cucurbita hybrid variety using shoot–tips of 5-day-old explants. The excised shoot–tips were cultured on Murashige and Skoog (MS) medium containing two plant growth regulators (6-benzyladenine and naphthaleneacetic acid) with various combinations and concentrations for the study of shoot induction. The best condition for shoot growth was with 3 mg l^–1 6-benzyladenine (BA) in MS medium. The shooting frequency was 84% and five shoots were obtained from each explant after 30 days of culture. Shoots (11.5 cm length) were rooted most effectively in 1 mg l^–1 indole-3 butyric acid (IBA)-supplemented MS medium. The highest root formation rate was 93% and all rooted shoots were transplanted into soil.     

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l^–1 gibberellic acid (GA₃) and 0.5 mg l^–1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l^–1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l^–1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l^–1-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.