In vitro storage ofXanthosoma spp. under minimal growth conditions

Xanthosoma germplasm requires regular subeulturing if stored in vitro under optimal culture conditions. An experiment was carried out to investigate whetherXanthosoma can be stored continuously under minimal growth conditions. Growth was reduced by incubation at low temperatures (9°C, 13°C and 17°C) and by adding mannitol (3.0% w/v) to the medium. Three investigatedXanthosoma species,Xanthosoma brasiliense, Xanthosoma robustum andXanthosoma sagittifolium could be stored in the dark for at least two years at 13°C. Addition of mannitol to the medium resulted in higher survival rates after three years of continuous storage. The storage period could be extended to at least 48 months forX. robustum andX. sagittifolium by annual subculture and regrowth under optimal culture conditions.     

In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4°C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.     

Plant regeneration in vitro of South Pacific taro (Colocasia esculenta var. esculenta cv. Akalomamale,Aracea)

Summary Axillary bud expiants from South Pacific (Solomon Islands) taro, Colocasia esculenta var. esculenta cv. Akalomamale (Araceae) cultured on a modified Murashige-Skoog medium containing 1 mg NAA 1^–1 and TE formed callus and produced multiple plantlets. Explants died if NAA was present at levels lower than 0.1 mg 1^–1. BA was not required and may have been inhibitory. Plantlets developed faster and became larger following transfer to a hormone-free medium two weeks after the start of culture. Fully grown plants were established in a potting mix and are growing well in a greenhouse.Abbreviations BA benzyladenine - BM basal medium - Ca Colocasia esculenta var. antiquorum - Ce Colocasia esculenta var. esculenta - Ck cytokinin(s) - CW coconut water - HSMSM half strength Murashige Skoog macroelements - HSMS half strength Murashige and Skoog medium - IM initial medium(ia) - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - SM second medium - TE taro corm extract - UCI University of California, Irvine     

In vitro storage of Eucalyptus grandis germplasm under minimal growth conditions

Slow growth-storage, for up to 10 months, has been achieved for Eucalyptus grandis shoot cultures by either the addition of 10 mg l⁻¹ abscisic acid to the growth medium or by the halving of nutrient supply (half MS) and removal of exogenous plant growth regulators. Reduction of light intensity or the addition of mannitol to the media were less effective in reducing growth rate. Isolated in vitro axillary buds encapsulated in calcium alginate and stored under low temperature and low light intensities survived for up to 3 months without loss in viability. Storage of such encapsulated fresh axillary buds at higher temperature resulted in a loss in viability. These methods have immediate applications to forestry breeding and clonal programs. This revised version was published online in June 2006 with corrections to the Cover Date.     

Variability in storage response within a coffee (Coffea spp.) core collection under slow growth conditions

Anin vitro core collection of African coffee germplasm, structured in 32 diploid diversity groups, was established and conserved under slow growth for 3 years (6 subcultures). The initial objective was to store twenty accessions per group, with four replicates per accession. A statistical model was developed to analyse observations of survival rates within each diversity group. The goodness of fit of the model was shown. Survival analysis indicated a broad variability of the accessions in their response to the storage conditions and confirmed the importance of structuring the coffee complex down to the intraspecific level. Intra- and inter-group differences had consequences on the genetic representativity of thein vitro core collection. For practical purposes, conservation was carried on when the intra-group genetic drift was less than 50%.Abbreviations BA 6-benzyladenine - CAR Central African Republic - CIRAD Centre de Coopération Internationale en Recherche Agronomique pour le Développement - FAO Food and Agriculture Organization - IBPGR International Board for Plant Genetic Resources - IDEFOR-DCC Institut Des Fôrets - Département Café Cacao - ORSTOM Institut français de recherche scientifique pour le développement en coopération     

Slow growth in vitro conservation of coffee (Coffea spp.)

The effects of reduced sucrose concentrations and low temperature on a collection of coffee microcuttings have been examined. Sucrose concentrations of 0.5 g l^-1 and 20 g l^-1 and temperatures of 20°C and 27°C were compared in three accessions: the Arabusta (interspecific hybrid) and Coffea arabica L. cv. Caturra amarillo and cv. Mokka de Tahiti. After six months, low sucrose concentrations reduced microcutting growth, rooting and survival rate. At 20°C, microcutting growth was also reduced, but leaf loss and survival rate were promoted. The genotypic differences at six months were minor. After one year without subculture, survival rate was influenced by sucrose concentration and by genotype. These two species can be cold-stored six months at 20°C on a medium containing at least 20 g l^-1 sucrose.Abbreviations BA 6-benzylaminopurine - MS Murashige & Skoog     

Effects of pre-storage conditions on storage of in vitro cultures of Nephrolepis exaltata (L.) Schott and Cordyline fruticosa (L.) A. Chev.

In vitro cultures of Nephrolepis exaltata and Cordyline fruticosa were stored at 5°, 9° or 13°C, at a low irradiance (3–5 mol m^–2 s^–1) or in darkness. Prior to storage the cultures were subjected to 18°, 21°, 24° or 27°C and 15, 30 or 45 mol m^–2 s^–1 in a factorial combination.The optimal storage conditions for Nephrolepis were 9°C in complete darkness. These cultures were still transferable to a peat/perlite mixture at the end of the experimental period of 36 months.The optimal storage conditions for Cordyline were 13°C and a low light level (±3–5 mol m^-2 s^-1). When the pre-storage conditions were normal growth room conditions (24°C and 30 mol m^-2 s^-1), in vitro cultures could be stored for 18 months. With the most favourable pre-storage treatment (18°C and 15 mol m^-2 s^-1) some cultures still had green shoots after 36 months of storage, but did not survive transfer to peat/perlite.Pre-conditioning before storage was most favourable for Nephrolepis, and not that important, but still favourable, for Cordyline. There was an interaction between pre-storage temperature and pre-storage irradiance. For both species a high irradiance level was less favourable than a low irradiance level when combined with high growth room temperatures.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - NOA 2-naphthoxyacetic acid     

Induction of callus from axillary buds of taro (Colocasia esculenta var. esculenta,Araceae) and subsequent plantlet regeneration

Summary Axillary buds of taro (Colocasia esculenta var. esculenta, Araceae) cultured on half strength Murashige-Skoog medium (HMS) containing taro extract (HMSTE) and 2, 4, 5-trichlorophenoxyacetic acid produce a compact, hard, slow growing callus which is not very active morphogenetically and produces only a few plantlets. When cultured on HMSTE plus 5 mg 1^–1 each of naphthaleneacetic acid and benzyl adenine (HMSNB) the buds produce a fast growing, friable and morphogenetically active callus. Meristematic regions form on the friable callus after 30 days on HMSNB. If transferred to HMSTE at this point the callus gives rise to plantlets. Addition of taro extract to the media is required for the culture of buds, induction of callus and plantlet regeneration.Abbreviations BA benzyl adenine - BNA b-naphthoxyacetic acid - CW coconut water (liquid endosperm) - DW dry weight - FW fresh weight - HMS half strength Murashige-Skoog medium - HMSCW HMSTE plus 100 ml CW 1^–1 - HMSNB HMSTE plus 5 mg 1^–1 each NAA and BA - HMSTE HMS plus 25 ml taro extract 1^–1 - HMSTR HMSTE plus 2 mg 2,4,5-T 1^–1 - MNA methyl-1-naphthaleneacetate - NAA naphthaleneacetic acid - OCPAA ortho-chlorophenoxyacetic acid - TE taro extract - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Effects of in vitro treatments on leaf area growth of potato transplants during acclimatisation

The importance of leaf area of in vitro propagated potato (Solanum tuberosum L.) plantlets for further growth during acclimatisation and the after-effects of in vitro treatments on growth were examined. The in vitro treatments included different levels of alar, nitrogen or mannitol or different temperatures during the last in vitro phase, the rooting phase. Leaf area or ground cover was recorded one day after planting to soil and at the end of the first phase of ex vitro growth, the acclimatisation phase. Regression analysis showed that leaf area of a transplant at the end of acclimatisation phase was positively influenced by leaf area of the same plantlet at the beginning of the phase. The relative increase in leaf area during acclimatisation (increase/early leaf area) was linearly related to the inverse of the early leaf area, indicating almost comparable relative increases for plantlets having larger early leaf areas, but more variable responses for plantlets having smaller early leaf areas. In vitro treatments mainly affected leaf area of transplants through their effects on early leaf area. Adding alar, reducing nitrogen and reducing temperature increased leaf area. Reducing mannitol increased ground cover. A lower nitrogen concentration and higher temperature in some cultivars had slight negative effects on the relative increase in leaf area after acclimatisation. For nitrogen these negative effects were less significant than the positive effects through early leaf area. Results stress the importance of manipulation of leaf area in vitro to enhance plant performance in later stages of growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Variability in storage potential of banana shoot cultures under medium term storage conditions

Shoot cultures of 401 banana clones were conserved under slow growth conditions (16±1°C, 25mol m^–2 s^–1). Storage duration-defined as 60% survival time of 20 shoot cultures of a clone-averaged 334 days. However, large differences occurred among the different genomic (sub)groups and even within the same (sub)group. East-African highland bananas and non-plantain AAB bananas can be stored for significantly longer periods. Shoot tip cultures of another 41 banana clones conserved at higher ambient temperature (22±3°C) needed to be subcultured sooner (every 220 days on average).Abbreviations BA 6-benzyladenine - CIRAD Centre de Coopération Internationale en Recherche Agronomique pour le Développement - IAA indole-3-acetic acid - IBPGR International Board for Plant Genetic Resources - INIBAP International Network for the Improvement of Banana and Plantain - PPF photosynthetic photon flux - QDPI Queensland Department of Primary Industries     

In vitro conservation of enset under slow-growth conditions

Studies on in vitro storage of enset under slow-growth conditions were carried out to develop an efficient protocol for conservation of the genetic diversity of the crop. The response to different growth retardation treatments was examined using three enset clones collected from southwestern Ethiopia. In vitro cultures could be effectively maintained for 6 months at 15 °C and 18 °C on MS medium supplemented with 10 μM BAP, in the presence of mannitol at concentrations of 0, 1 or 2% as a growth retardant. Shoots were subsequently recovered and multiplied on MS medium supplemented with 10 and 20 μM BAP at 25 °C and rooted shoots were successfully transferred to the greenhouse. Incubation at the lower temperature (15 °C) and the presence of mannitol in the culture medium had a significantly positive effect on maintenance, measured by the number of recovered shoots after storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic diversity in Taro (Colocasia esculenta Schott, Araceae) in China: An ethnobotanical and genetic approach

Taro,Colocasia esculenta (Araceae), is a widely distributed and important food crop in the humid tropics and subtropics. Relatively neglected by science, much knowledge of genetic diversity in taro is with farmers. Taro genetic resources managed by five ethnic communities and Han farming villages in diverse ecosystems were sampled and characterized in Yunnan Province, southwest China. This study documented a new type of flowering taro selected by farmers which is widely and intensively cultivated for its edible flower. Samples representing 20 traditional cultivars were grouped into five major morphotypes according to ethnobotanical, agro-morphological, and preliminary genetic characterization. Folk taxonomy and uses tended to confirm the five morphotypes recognized by peoples of Yunnan for their distinctive properties and uses. These major taro morphotypes are key units for assessing how patterns of use maintain genetic diversity and to monitor potential genetic erosion. The morphotype groups also suggest possible evolutionary relationships in cultivated taros.     

Effect ofin vitro storage at 4°C on survival and proliferation of two apple rootstocks

One cm long shoot explants of dwarf apple rootstocks P 2 and M.9 taken from 2 year-old cultures were stored at 4°C in the dark in three media differing in concentration of growth regulators. Every 6 weeks, some explants were transferred into proliferation medium and multiplication rate was observed during three or four consecutive passages. In a second experiment, the influence of explant type (1 cm long shoot tips, 1 cm long middle part of shoots or three-shoot tufts smaller than 1 cm) and transfer time to the cold room (immediately, 10 days, or 20 days after subculture) on explant survival and proliferation were analysed.Survival of explants was influenced by composition of the storage media. On medium without 6-benzylaminopurine, 70% of P 2 and 17% of M.9 explants became necrotic during 18 weeks of storage. P 2 rootstock proliferated better in three passages after storage than did unstored controls. Storage of M.9 rootstock reduced proliferation in the first and second passages if stored in media containing 6-benzylaminopurine in comparison with unstored controls. Explants stored as tufts and transferred to the cold room directly after subculture produced more shoots during two passages than cultures stored as single shoots.     

Sucrose in storage media and cultivar affects post-storage regrowth of <Emphasis Type="Italic">in vitro</Emphasis> Hosta propagules

The goal of this research was to investigate if culturing in high sucrose (5%) liquid media during multiplication phase (stage II) would enhance endogenous sugar levels and dry matter sufficiently to allow storage of in vitro plants in sugar free media without adversely affecting post-storage recovery. Hosta tokudama Newberry Gold (NBG) and Hosta Striptease were cultured in Murashige and Skoog (MS) media containing 5% sucrose during stage II and transferred to rooting phase (stage III) in MS medium without (0%) sucrose or with 3% sucrose for 4weeks. At the end of stage III, cultures were stored, with the remaining media, at 10°C with 5molm^–2s^–1 photosynthetic photon flux (PPF) from cool white fluorescent lamps for 7 or 14weeks with or without a 2-week dark period prior to removal from storage. In both cultivars, stage III plants cultured in 3% sucrose media had higher soluble sugar levels and greater shoot and root biomass than those cultured in 0% sucrose media. Shoot and root soluble sugars decreased during storage. Shoot growth ceased during storage in both media. Root dry matter continued to increase in plants stored in 3% sucrose media but did not change in 0% sucrose media. Plants cultured in 3% sucrose media had less leaf chlorosis and less mortality after 7 or 14weeks of low temperature storage than the plantlets from sugar free media. Extending the storage period from 7 to 14weeks or introduction of 2-week dark period at the end of storage did not affect leaf chlorosis or plant mortality during acclimatization. Post-storage growth varied with the cultivar. Benefit of having sucrose in storage media was to develop a strong root system that aided the acclimatization and post-storage growth following 7 or 14week storage. Sucrose loading by culturing plants in liquid media containing 5% sucrose did not allow storage in sugar free media without adversely affecting post-storage growth in both cultivars.     

Effect of various growth regulators on growth in vitro of cherry shoot tips

An investigation was undertaken to examine the effects of nine different growth regulators on growth in vitro of shoot cultures of the semi-dwarfing cherry rootstock Colt (Prunus avium × P. pseudocerasus). The effects of each supplement on shoot extension and proliferation and also leaf and callus production were noted, and it was found that BAP has the ability to proliferate shoots, IBA nullifies this effect and that kinetin, ABA, GA₃ and ethylene inhibit the growth of colt cultures. Conditions were established which resulted in a) optimum shoot growth prior to subsequent rooting or grafting; b) maximum shoot proliferation for rapid clonal multiplication and c) minimum shoot growth. This study will form the basis of an investigation into germplasm conservation of temperate fruit trees by both cryogenic storage and minimal growth techniques.     

Growing of potato microplants in the presence of alginate-silverthiosulfate capsules reduces ethylene-induced culture abnormalities during minimal growth conservation in vitro

Alginate capsules containing anionic complex silverthiosulfate (STS) [Ag(S₂O₃)₂ ^3-] were placed in the culture tubes over minimal growth media for studying whether STS could be used at higher concentrations to sustain ethylene-inhibiting effect during conservation of microplants in six potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of STS (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 mM) were incorporated into the alginate capsules, and 12 alginate-STS capsules were tested in semisolid (7 g l^–1 agar) minimal growth medium containing 20 g l^–1 mannitol and 40 g l^–1 sucrose. This indirect supplementation of STS through alginate capsules rendered reduced total availability of STS in the minimal growth medium as compared to when it was directly supplemented in the medium at a given concentration. Growing of microplants in the presence of alginate-STS capsules improved the microplant growth and reduced the culture abnormalities over a period of 16 months under minimal growth conditions. Most significant improvement in microplant growth was in terms of green leaf production and leaf senescence. Vitrification, flaccidity and other growth abnormalities, viz., leaf loss, abnormal stem swelling and necrosis were not observed when the microplants were conserved in the presence of alginate-STS capsules. To foster optimum microplant growth and reduce culture abnormalities, potato microplants could favourably be maintained in the presence of 0.5–1.0 mM alginate-STS capsules during minimal growth conservation. Higher concentrations of alginate-STS capsules (>1.0 mM) were in general detrimental to potato microplant growth and survival during prolonged storage in vitro. Release kinetics of STS from the alginate-STS capsules, its distribution in the medium and accumulation of silver in potato microplants were studied using ^110mAg. The release rate of STS from the capsules was found to be directly proportional to the concentrations of alginate-STS capsules. A distinct concentration gradient of ^110mAg in the medium with increasing depth from top to bottom, and its accumulation in the potato microplants may be attributed to the improved anti-ethylene action of STS at higher concentrations through alginate capsules.     

Responses of strawberry plantlets cultured in vitro under superbright red and blue light-emitting diodes (LEDs)

Unrooted strawberry cv. `Akihime' shoots with three leaves obtained from standard mixotrophic cultures were cultured in the ``Culture Pack'-rockwool system with sugar-free MS medium under CO<sub>2</sub>-enriched condition. To examine the effect of superbright red and blue light-emitting diodes (LEDs) on in vitro growth of plantlets, these cultures were placed in an incubator, ``LED PACK', with either red LEDs, red LEDs1blue LEDs or blue LEDs light source. To clarify the optimum blue and red LED ratio, cultures were placed in ``LED PACK 3' under LED light source with either 100, 90, 80, or 70% red + 0, 10, 20, 30% blue, respectively, and also under standard heterotrophic conditions. To determine the effects of irradiation level, cultures were grown under 90% red LEDs + 10% blue LEDs at 45, 60 or 75 mol m^–2 s^–1 . Plantlet growth was best at 70% red + 30% blue LEDs. The optimal light intensity was 60 mol m^–2 s^–1. Growth after transfer to soil was also best after in vitro culture with plantlets produced were 70% red LEDs + 30% blue LEDs.     

Amino acid sequences of ferredoxin isoproteins from Japanese taro (Colocasia esculenta Schott)

The amino acid sequences of ferredoxins (Fd A and Fd B) from Japanese taro (Colocasia esculenta Schott) were determined. They consisted of single polypeptide chains of 98 residues, and both Fds had molecular masses of 10700 and 10500, respectively. There was a 92% homology between the sequences of the isoproteins (Fd A and Fd B). These sequences were compared with those of the closely related plant Fds and their phylogenetic tree was constructed. Two ferredoxin isoproteins from Hawaiian taro (Colocasia esculenta Schott) were also isolated and their N-terminal sequences were determined to be identical to those of Japanese taro.     

In vitro propagation of taro,with spermine,arginine, and ornithine

In vitro growth and multiplication of taro [Colocasia esculenta var. antiquorum cv. Keladi Birah] was improved considerably, when primary shoot apices were cultured on two modifications of Linsmaier and Skoog [1965] medium, containing 5.5 mg 1^–1 naphthaleneacetic acid and 0.2 mg 1^–1 kinetin or 1.85 mg 1^–1 naphthaleneacetic acid and 2 mg 1^–1 kinetin and supplemented with 10^–4 or 10^–3 mol·1^–1 of polyamine spermine or either of the precursors of polyamine putrescine—arginine and ornithine. Plantlets were regenerated directly from primary shoot apices, axillary buds and protocorm-like bodies [PLB]. Frequency of plantlet regeneration, rate of development and growth in height of main plantlets were enhanced by the addition of arginine and ornithine to the media. Secondary plantlet formation from axillary buds and PLB were promoted by spermine and arginine respectively.