Slow-growth conservation of potato microplants: efficacy of ancymidol for long-term storage in vitro

Ancymidol was investigated as an alternative mediumsupplement to mannitol for slow-growth conservation ofpotato microplants in vitro. Differentconcentrations of ancymidol (0, 5, 10, 15, 20, 25, 30,35 and 40 M) were tested in slow-growthmedia based on MS medium supplemented with either 30or 60 gl^-1 sucrose. The cultures were conservedunder a 16-h photoperiod at two temperature regimesi.e. 24 ± 1 ^°C and 6 ± 1 ^°C. Therewere significant interactions between ancymidol andother factors such as sucrose, temperature andgenotype for microplant survival, microshoot heightand overall microplant growth. Ancymidol did have abeneficial effect on culture viability after prolongedmaintenance in vitro. The growth-inhibitingeffect of ancymidol persisted through a 16-monthculture period. Combined effect of ancymidol, sucroseand temperature showed that optimum culture viabilityand desirable microplant growth were obtained when thecultures were grown in MS medium supplemented with 10M ancymidol plus 60 gl^-1 sucrose at6 ± 1 ^°C. Vitrification and flaccidity, whichare very frequently observed in potato microplantcultures during prolonged maintenance in vitrounder osmotic stress (mannitol), were not observedwhen the microplants were conserved in ancymidolmedia. Genetic stability of potato microplantsconserved in ancymidol media was evaluated usingrandomly amplified polymorphic DNA (RAPD)fingerprints. Ancymidol did not induce any detectablegenetic variation in genomic DNA as visualized by theabsence of either any additional RAPD fragment oralterations in RAPD fragment patterns.     

Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.     

Resynthesizing <Emphasis Type="Italic">Brassica napus</Emphasis> from interspecific hybridization between <Emphasis Type="Italic">Brassica rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis> through ovary culture

Using three varieties of Brassica rapa, cv. Hauarad (accession 708), cv. Maoshan-3 (714) and cv. Youbai (715), as the maternal plants and one variety of B. oleracea cv. Jingfeng-1 (6012) as the paternal plant, crosses were made to produce interspecific hybrids through ovary culture techniques. A better response of seed formation was observed when ovaries were cultured in vitro at 9–12 days after pollination on the basal MS and B5 media supplemented with 6-benzylaminopurine (BA) and naphthylacetic acid (NAA). The best response was observed for cross 714×6012 with the rate of seeds per ovary reaching 43.0%. Seeds for cross 715×6012 showed the best germination response (66.7%) on the regeneration medium (MS+1.0 mg l^–1 BA+0.05 mg l^–1 NAA). In all three cross combinations, good response in terms of root number and length of plants was observed on the root induction medium (MS+1.0 mg l^–1 BA+0.1 mg l^–1 NAA). A better response was observed for the regenerated plants cultured for 14 days than for 7 days. The ovary-derived plants with well-developed root system were hardened for 8 days and their survival rate reached over 80%. Cytological studies showed that the chromosome number of all plants tested was 19 (the sum of both parents), indicating that these regenerated plants were all true hybrids of B. rapa (n = 10) × B. oleracea (n = 9). The regenerated plants were doubled with colchicine treatment, and the best response in the crosses 708×6012, 714×6012 and 715×6012 was observed when treated with 170 mg l^–1 colchicine for up to 30 h and their doubling frequency reached 52, 56 and 62%, respectively.     

Somatic cell genetic studies inBrassica species. II. Production of androgenetic haploid plants inBrassica nigra (L.)Koch

Summary Callus growth and its subsequent regeneration into complete plantlets was achieved from in vitro cultured anthers ofBrassica nigra (L.)Koch. Callus was induced on a modified N6 medium containing trace elements, organics of B5 medium and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Morphogenesis of callus in the form of shoots on MS medium containing indole-3-acetic acid (IAA) and N⁶-benzyladenine (BA) 0.5 mg/l each and embryoids on MS medium containing 0.5–1.0 mg/l IAA and 3.0–5.0 mg/l BA could be accomplished. Chromosomal analysis revealed presence of 41% haploids (n=8) amongst the regenerated plants.     

Production of interspecific hybrids between Alstroemeria pelegrina L. var. rosea and A. magenta Bayer by ovule culture

Interspecific hybrids were efficiently produced in the cross-incompatible combination between Alstroemeria pelegrina L. var. rosea and A. magenta Bayer by culturing immature ovules with placenta 7–14 days after pollination on 2 g/l Gelrite-solidified MS medium containing 3% (w/v)sucrose. The plants showed intermediate characteristics between the parents and their hybridity was confirmed by karyotype and DNA analyses. The mean number of chromosome association per PMC at metaphase I was 2.60I+6.70II, pollen stainability was20.8%, and they produced viable seeds after self-pollination. Furthermore, mature plants were obtained when the hybrids were backcrossed as male parents with both the parents. The backcross-progeny from A. pelegrina var. rosea × hybrids exhibited 3.8 to 79.7% pollen stainability and that from A. magenta × hybrids 78.8 to 98.3%. Almost all of these plants produced viable seeds after self-pollination, which implies that they can beutilized for breeding of novel cultivars of Alstroemeria. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of Chinese Spring regenerants obtained from short- and long-term wheat somatic embryogenesis

Summary Somatic embryogenesis was initiated from immature embryos on Murashige-Skoog (MS) medium plus 2 mg.l^-1 2,4-dichlorophenoxyacetic acid, 2% sucrose and 0.6% agarose. Somatic embryos were isolated and regenerated into whole green plants on MS medium devoid of 2,4-D. These regenerants were previously demonstrated to differ in their mitochondrial DNA organization. In order to estimate their characteristics three progenies of short-term culture regenerants and three progenies of long-term culture regenerants were analyzed and compared to the parental line. These somaclones obtained from the wheat variety Chinese Spring were evaluated for variation of 13 agronomic and morphological quantitative characters in comparison to the parental line. Significant variation was observed for plant height, spike length, main tiller diameter, between the somaclones regenerated from long-term culture and their parent. Differences were observed to increase with the duration of culture, leading to a significant modification of the structure of the plants. Several changes occurred during the somatic tissue cultures, but to a lesser extent than has previously been described in the literature.     

Plant regeneration via organogenesis and somatic embryogenesis in callus cultures derived from mature zygotic embryos of leek (Allium ampeloprasum L.)

Summary A high frequency plant regeneration system via organogenesis and somatic embryogenesis was established with callus cultures derived from mature zygotic embryos of different leek genotypes (Allium ampeloprasum L.). Four different callus types with varying morphogenetic potential were obtained. Relatively high concentrations of the auxin 2,4-dichlorophenoxy-acetic acid reduced callus weight and subsequent shoot regeneration and primordia formation of the callus. Shoot regeneration and primordia formation of the callus decreased after prolonged subculture on media containing 2,4-dichlorophenoxy acetic acid. A callus growth period of six weeks on Murashige and Skoog medium with 0.25–0.5 mg l^-1 2,4-dichlorophenoxy acetic acid showed the highest rate of shoot regeneration after transfer of callus to regeneration medium with 1 mg l^-1 kinetin.Differences between leek genotypes in callus type, callus weight, shoot regeneration and primordia formation were observed. Histological observations showed that plant regeneration took place, both via the pathway of somatic embryogenesis and organogenesis.Abbreviation 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog (1962) medium     

Plant recovery of cryopreserved apical meristem-tips of Melia azedarach L. using encapsulation/dehydration and assessment of their genetic stability

A cryopreservation process usingencapsulation/dehydration technique was setup for apical meristem-tips of invitro plantlets of `paradise tree' (Melia azedarach L. var. gigantea,clone `El dorado'). Apical meristem-tipswere cultured for one day on MS basalmedium with 2 M BA and 0.5 M IBAand encapsulated with 3% sodium alginate.The highest shoot proliferation rate aftercryopreservation was obtained whenencapsulated apical meristem-tips werepregrown for 3 days in liquid medium with0.5, 0.75 and 1 M of sucrose for 24 hoursprogressively, desiccated for 5 hours withsilicagel followed by rapid or slowcooling. Survival after freezing in liquidnitrogen ranged between 67–83% andshoot proliferation ranged between 43–60%. This cryopreservation treatmentpreserved genetic stability, when it wasevaluated using the electrophoreticpatterns of nine isozyme systems and RAPDprofiles.     

Somatic embryogenesis and plant regeneration of Tripsacum dactyloides L.

Summary This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 mol or 20 mol) and sucrose (3% or 6%), and plants were regenerated on hormone free MS medium containing 2% sucrose. No significant differences were found in callus initiation frequency or in embryogenic response of cultures on the four combinations of sucrose and dicamba tested. The embryogenic cultures have been maintained for 9 months (12 subcultures) and have retained regeneration capacity. Plants regenerated from tissue culture of maize-by-Tripsacum hybrids could be useful in maize improvement.     

Induction and development of adventitious shoots of Atropa baetica as a means of propagation

In vitro propagation of Atropa baetica was established employing axillary buds. Single buds were cultured through a multiple shoot induction phase, rooting phase, and then followed by acclimatization in soil. For multiple shoot induction, Murashige and Skoog (MS) medium with 3% sucrose, supplemented with either 0.75 or 1.25 mg l^-1 of BAP provided the best results with an average of 5.6 shoots per explant after 31 days of culture. Similar results were obtained with higher BAP concentrations (1.75–2.0 mg l^-1); however, these media had a negative effect on the subsequent root induction due to residual BAP effect. Medium containing only 0.25 mg l^-1 of BAP induced a significantly lower number of shoots. Root induction occurred spontaneously after transferring the shoots onto MS medium lacking any plant growth regulator. Moreover, root induction also occurred on media supplemented with 0.125 and 0.25 mg l^-1 of NAA. On these two rooting media, this response was more prominent and with a higher number of roots per explant. Nevertheless, after 28 days on root induction medium, the number of rooted plantlets was similar on the three media. Acclimatization of plantlets in soil was very successful (95.52%). However, all plantlets which died during acclimatization were rooted on medium containing 0.25 mg l^-1 NAA suggesting a negative carry over effect of this medium upon plantlet survival, irrespective of the initial BAP treatment used. On the other hand, karyological studies showed no variation in the number of chromosome (2n=72) in root tips of the plantlets produced. This revised version was published online in July 2006 with corrections to the Cover Date.     

Clonal propagation of Acacia saligna by shoot tip culture

Summary Multiple shoots buds were obtained successfully from shoot tips of Acacia saligna by placing explants into solidified Murashighe & Skoog (1962) medium (MS medium) supplemented with 5.0 to 9.0 mg/L BAP. Sequential culture treatment was highly effective for shoot elongation using MS medium containing 0.3 mg/L BAP and 0.2 mg/L IAA. The shoots rooted best on MS medium supplemented with 2.0 mg/L IBA. Plantlet survival after transfer to soil was more than 90%. The shoot proliferation method described could be used for the mass clonal propagation of selected genotypes.     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

Factors affecting minimal growth conservation of potato microplants in vitro

Combined effects of sucrose, mannitol and photoperiod on microplant conservation were studied in four potato genotypes belonging to two different groups viz., Tuberosum and Andigena. Minimal growth medium was based on Murashige and Skoog (MS) supplemented with 6 different concentrations of sucrose (30, 40, 50, 60, 70 and 80 gl^-1) with 4 different concentrations of mannitol (0, 20, 40 and 60 gl^-1). The cultures were conserved under two photoperiod conditions i.e. continuous illumination and 16-h photoperiod at 6 ± 1 °C. There were significant interactions between photoperiod and sucrose, and between photoperiod and mannitol. Maximum microplant survival and desirable microplant growth were observed under 16-h photoperiod. Sucrose alone did not improve culture viability over 30 months of storage. Inclusion of mannitol in the conservation medium increased microplant survival. Sucrose x mannitol interaction showed that sucrose was effective in enhancing microplant survival in combination with 20 or 40 gl^-1 mannitol, but not with 60 gl^-1 mannitol. Combined effect of sucrose, mannitol and photoperiod showed that optimum microplant growth and maximum culture viability were obtained when the cultures were grown in MS medium containing 40 gl^-1 sucrose and 20 gl^-1 mannitol under 16-h photoperiod. Potato microplants can be conserved in this medium and cultural conditions up to 30 months without subculturing. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic factors influencing the regeneration ability of rye (Secale cereale L.). II. Immature embryos

Summary Immature embryos of seven rye inbred lines were cultured on modified MS medium containing 3 mg/dm^–3 2,4-D. According to thein vitro response lines were divided into four groups: (1) those producing non-embryogenic callus (NEC) from above 30% of the embryos and having a high rate of non-responding (NR) embryos, (2) those producing non-embryogenic callus (NEC) from the majority of embryos, (3) those producing NEC by the majority of embryos with a high percentage of calli regenerating roots, (4) those producing embryogenic callus (EC) and regenerating plants by above 50% of the embryos. The inheritance of these response types was analysed in F₁, F₂, and F₃ generations of crosses of some lines. The results obtained indicate that EC production and both plant and root regeneration are determined by recessive genes whereas the reduced ability for NEC production most probably by dominant genes. The lack of response is controlled by at least two interacting genes.     

Plant Regeneration from in vitro Cultured Anthers of Black Mustard (Brassica nigra Koch)

Anthers of Brassica nigra, excised from fresh as well as cold-pretreated (3 days at 3 ± 2°C) buds cultivated on modified B₅ medium (Gamborg et al. 1968) containing sucrose level varying from 2 % to 10 %, along with 1O^?6M BAP (benzylaminopurine) and 9 × 10^?6M 2,4-D (2,4-dichlorophenoxyacetic acid), developed calli and/or embryos. The latter response was observed only in anthers reared on media containing 6 % or higher levels of sucrose. On media containing two or four per cent sucrose, the anthers produced calli, exclusively. The growth of embryos was inhibited or else they started callusing if left on the media containing higher levels of sucrose. However, on transfer to MS medium (Murashige and Skoog 1962), containing 2 % sucrose, embryos started callusing and subsequently a few secondary embryos differentiated. Such embryos were sub-cultured on MS + 5 × 10^?6M BAP + 2 % sucrose, wherein numerous shoots developed from embryos. The shoots were rooted by transferring to a medium containing 5 × 10^?6M NAA (naphthalene acetic acid). Within two months of culture, some of these plants started flowering in vitro.     

In vitro propagation of Japanese garden iris,Iris ensata Thunb.

Summary Explants of young scapes of Iris ensata were cultured on MS medium with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar, and this species was characterized by high variety specificity for callus, shoot and root induction. Among 23 varieties and one wild form tested, Okichidori, Miyukisudare and Meiji-l exhibited a considerable rate of shoot induction, although these induced poorly rooted shoots. In addition, two types of callus induction such as green and white calli were observed, and the induction of green-type calli was significantly correlated with that of shoots. Surprisingly, the only modification, half-strength MS inorganic salts, for the above medium proved to be very effective for shoot induction in the scape culture. For shoots obtained from the scape culture, effects of sucrose concentrations and activated charcoal on root induction were examined by using 1/2 MS with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar as the basic medium. The addition of 1% activated charcoal to the media had a marked effect for root induction independent of sucrose concentrations and varieties tested. The in vitro propagation technique of I. ensata is discussed.     

Factors affecting in vitro flowering and fruiting of green pea (Pisum sativum L.)

Multiple shoots were efficiently regenerated from cotyledonary node and shoot tip explants of Pisum sativum within 15 days on MS medium containing B₅ vitamins and supplelmented with 2.0 mgl^-1 6-benzylaminopurine. The elongated shoots produced on the same medium were excised and transferred to MS medium containing half strength ammonium nitrate (8.25 gml^-1) and supplemented with auxins (indole-3-butyric acid or naphthalene acetic acid) either alone or in combinations with gibberellic acid. Rooting and flowering were observed on the 7th and 15th day after their transfer to rooting medium. The flowers self-fertilised in vitro and produced mature pods within 25 days of rooting. These seeds were germinable both in vitro and in vivo. In vitro seeds sown in pots under field conditions developed into flowering plants, and subsequently produced pods with viable seeds. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improved plant heterokaryon formation by surfactant-supplementation of polyethylene glycol fusogen solution

Summary PEG fusion solution for leaf protoplasts of Petunia parodii and cell suspension protoplasts of albino P. hybrida cv. Comanche was supplemented with 0.01–1.0% (w/v) Pluronic F-68. This stimulated protoplast fusion overall, including parental homokaryon formation, with increased means of 23% and 83% respectively, over appropriate controls using 1.0% (w/v) surfactant added to the standard PEG solution. Interestingly, the percentage heterokaryon formation increased near 2-fold (P<0.001) for fusogen solutions supplemented with 0.01% (w/v) Pluronic. Protoplasts regenerated to colonies in KM8P/KM8 liquid medium, indicating no adverse effects of Pluronic F-68 on viability, both in the short and longer terms.Abbreviations BA 6-benzyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - PEG polyethylene glycol     

High frequency direct plant regeneration from leaf and petals of Cape Primrose (<Emphasis Type="Italic">Streptocarpus</Emphasis>)

High frequency direct plant regeneration from leaf and petal explants was accomplished for the first time in Streptocarpus varieties. The shoot induction frequency varied with respect to the benzylaminopurine (BAP) concentration added to the Murashige and Skoog (MS) medium. MS medium with 0.5 mg l⁻¹ BAP exhibited the highest (69.9%) plant regeneration frequency with an average of 186 shoots per explant. A higher concentration of BAP inhibited shoot bud induction and plant regeneration along with necrosis of explants. Petal explants derived from the varieties ‘Branwen’ (pink and white) and ‘Chorus Line’ (violet and white) displayed plant regeneration frequency of 22.2–47.4% (within a total of 12 weeks) on MS medium containing 2.0 mg l⁻¹ α-naphthaleneacetic acid and 0.5 mg l⁻¹ BAP for 8 weeks followed by 4 weeks on MS medium with 1.0 mg l⁻¹ BAP. Scanning electron microscopy confirmed direct plant regeneration without callus. Regenerated plants from leaf explants with well-developed leaves and roots were hardened and successfully transferred to pots in glasshouse exhibiting 86% survival at the end of 4–6 weeks. Whereas, regenerated plants from flower petal explants upon transfer to pots in glasshouse exhibited 75–82% survival at the end of 4–6 weeks.     

Inter-sectional hybrids obtained from reciprocal crosses between Begonia semperflorens (section Begonia) and B. ‘Orange Rubra’ (section Gaerdita × section Pritzelia)

Inter-sectional hybrids were successfully obtained by the reciprocal crosses between 11 cultivars (including 6 diploids and 5 tetraploids) of Begonia semperflorens (SS & SSSS genomes) and B. ‘Orange Rubra’ (RR genome) with the aid of in vitro culture of mature or immature seeds on MS medium containing 0.1 mg l⁻¹ α-naphthylacetic acid, 0.1 mg l⁻¹ 6-benzyladenine, 10 mg l⁻¹ gibberellic acid, 30 g l⁻¹ sucrose and 2.5 g l⁻¹ gellan gum. Embryo rescue as ovary culture with immature seeds 12^th–16^th day after pollination (DAP) generally gave higher efficiency of plantlet formation, but in some cross combinations, culture of mature seeds (30 DAP) resulted in higher yield of plantlets. Flow cytometric analysis revealed that they were consisted of the plants with various genomic combinations (RS, RR, RSS, RRS, RRSS and RRRRSS) as estimated by the DNA contents of both parents. Hybridity of these plants with various genomic combinations including RR was confirmed by random amplified polymorphic DNA analysis. These results suggested that unreduced gamete formation and spontaneous chromosome doubling were involved in the hybrid formation of various ploidy levels and genomic combinations. These hybrids showed various levels of intermediate traits between both parents according to the genomic compositions, and some of them had desirable characters of both parents.