In vitro bulblet production of Lachenalia

In vitro bulblet formation and subsequent transplanting of bulblets to soil were studied in order to develop a cost-effective method for the mass production of three Lachenalia varieties. Clumps of adventitious shoots regenerated from leaf explants were used. Bulblet formation was initiated after 2 weeks when shoots were subjected to low temperature (4–15 °C). The size (age) of the adventitious shoot affected the bulblet size, and shoots shorter than 4 mm did not form bulblets. Larger bulblets formed on medium containing 6% sucrose compared to 3% sucrose. Following bulblet initiation, illumination was not necessary for the completion of bulblet formation. Bulblets went into dormancy 3–4 months after they had been initiated or when the culture medium dried out, and they were released from dormancy when the natural night temperatures started to decrease in the late summer. The survival rate of the bulblets after transplanting was directly correlated to the size of the bulblets.The most important factors influencing in vitro bulblet formation of Lachenalia were sucrose concentration, temperature and length of explant shoots. Received: 12 June 1998 / Revision received: 8 September 1998 / Accepted: 23 September 1998     

In vitro propagation from axenic explants of <Emphasis Type="Italic">Lilium oxypetalum</Emphasis> (D. Don) Baker,an endemic bulbous plant of high altitude Himalaya

In vitro propagation protocol for Lilium oxypetalum, a high altitude Himalayan endemic lily, has been developed. Effect of explant types (i.e., callus and in vitro bulblet scales) and sucrose concentration [0–6.0% (w/v)] on in vitro bulblet regeneration of L. oxypetalum was tested in previously optimized Murashige and Skoog basal medium supplemented with 2.0 μM 6-benzyladenine and 0.1 μM α-naphthaleneacetic acid. Callus explants produced significantly (P < 0.01) higher number of bulblets per explant than bulblet scale explants. Of the different concentrations of sucrose tested, 4.5% (w/v) sucrose showed significantly (P < 0.01) higher percentage regeneration (i.e., 70.8 ± 4.2 and 79.2 ± 4.2% regeneration on callus and bulblet scale explants, respectively), and produced higher number of bulblets per explant (i.e., 9.0 ± 0.4 and 5.4 ± 0.5 bulblets on callus and bulblet scale explants, respectively). Regenerated bulblets developed 2–3 leaves when subcultured for 4 weeks and were subsequently transferred ex vitro with a survival rate of 66.7% after 6 weeks. Leaves of the survived plantlets became dry after growing ex vitro for 10 weeks, amongst which 86.4% re-sprouted after remaining dormant for 5–6 weeks and produced 1.5 bulblets per explant. Findings of the present study hold promise for efficiently multiplying the target species in view of its potential economic and conservation significance.     

High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures

In vitro shoot proliferation and bulblet production of garlic (Allium sativum L.) was studied in liquid cultures. Shoots grown in vitro were used as explants and were cultured in MS medium supplemented with 2% (w/v) sucrose and 0.5 mg l^–1 2-iP. Three culture methods (semi-solid, liquid-immersion and raft) were compared for shoot proliferation. Explants in liquid (immersion) culture exhibited an increased multiplication rate and fresh weight of shoots after 3 weeks of culture as compared with the other treatments. Bulblet formation and growth were studied in liquid medium with different concentrations of sucrose (2–13%). MS medium containing 11% (w/v) sucrose was optimal for bulblet development and bulblets developed in this medium within 9 weeks in culture. The highest multiplication rate was (135 bulblets/explant) found when explants were cultured in bulbing medium (MS medium containing 0.1 mg l^–1 NAA+11% (w/v) sucrose) supplemented with 10 M JA. Growth retardants CCC, B-9, ABA also promoted induction and growth of bulblets. Darkness promoted the bulblet induction and growth compared to light conditions (16-h photoperiod of 50 mol m^–2 s^–1). The dormancy of bulblets was broken by cold treatment at 4 °C for 8 weeks.     

In vitro bulb development in shallot (Allium cepa L. Aggregatum Group): effects of anti-gibberellins,sucrose and light

Bulbing was studied in shallot plants cultured in vitro. Bulbing occurred under a 16 h photoperiod with fluorescent + incandescent light and 30-50 g 1(-1) sucrose in the culture medium. Exogenous gibberellin (10 microM GA3) inhibited leaf and root growth and bulbing. When added to the medium at a concentration of 10 microM, three inhibitors of gibberellin biosynthesis (ancymidol, flurprimidol and paclobutrazol) promoted bulb formation and the percentage of bulbing. When ancymidol was used in combination with GA3, it did not reverse the effect of GA3 applied alone. Under treatments with 30-70 g l(-1) sucrose, bulbing ratios greater than those found in control plants were achieved by addition of ancymidol, and bulb fresh weight was increased in the same way. Ancymidol caused a 66% decrease in sucrose content in leaf bases but greatly increased the glucose, fructose and fructan contents. The increase in fructan content by ancymidol could result from the three-fold rise in total [14C]sucrose uptake per plant from the culture medium associated with a marked increase in leaf base labelling at the expense of root labelling. The possible role of ancymidol is discussed and evidence supports a major regulatory role for gibberellins in bulbing.     

Endogenous hormone levels in habituated nucellar Citrus callus during the initial stages of regeneration

 Embryogenic nucellar callus cultures of different Citrus species and cultivars growing in hormone-free medium were transferred to medium containing either sucrose or glycerol as the only carbohydrate source. Glycerol has been reported to induce further development of Citrus somatic embryos, while in the presence of sucrose they continue to proliferate in an 'undifferentiated' manner. The endogenous hormone levels of the cultures were evaluated after 2 and 5  days to characterise the initial steps of embryo development. In most cases, differences among treatments were observed only after 5 days of culture. Higher cytokinin levels were found in most of the cultures transferred to the glycerol-containing medium. The effect of ageing sweet orange cultures on their endogenous hormone levels was determined by leaving them in the original culture medium without subculturing for 60 days. While no changes were observed in the free indoleacetic acid and gibberellin contents, lower levels of abscisic acid and cytokinins were found in the aged cultures than in those transferred at the normal interval, every 30 days. The endogenous hormone contents of Citrus callus of different genotypes were compared. Significant differences were observed in the levels of all hormones evaluated, even when the in vitro ontogeny of the different genotypes was very similar. Received: 10 February 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Physiological and biochemical responses of tomato microshoots to induced salinity stress with associated ethylene accumulation

Physiological and biochemical responses of open-pollinated ‘Roma’ and dwarf F1 hybrid ‘Patio’ tomato (Lycopersicon esculentum Mill.) cultivars to in vitro induced salinity were examined in light of the possible contribution of ethylene to these symptoms. Salinity was induced by incorporating 0 (control), 50, 100, 150, or 200 mM NaCl into shoot culture media. Elevated salinity treatments significantly enhanced ethylene accumulation in the headspace and were accompanied by increased leaf epinasty in both cultivars. Growth, leaf cell sap osmolarity, leaf tissue viability and shoot soluble protein content were generally depressed with elevated salinity treatments, whereas electrolyte leakage, membrane injury, raffinose, and total sugars were concomitantly increased. Macronutrients N, P, K, Ca, Mg, and S decreased with elevated salinity in both cultivars and were accompanied by a significant increase in Na content and a sharp decrease in K/Na ratio. Tissue micronutrients, Fe, B, Zn, Mn, and Cu were generally decreased with elevated salinity especially at 100 mM or more. Incorporating ethylene inhibitors CoCl₂ or NiCl₂ at 5.0 or 10.0 mg/l into media supplemented with 100 mM NaCl significantly reduced ethylene accumulation in the headspace and prevented epinasty, but did not eliminate the negative impacts on growth and other physiological parameters caused by salinity treatment in either cultivar. Our results indicate that the increase in ethylene under salinity stress is not the primary factor contributing to salinity’s deleterious effect on tomato plant growth and physiology.     

Induction of somatic embryogenesis and in vitro flowering from inflorescences of chamomile (Chamomilla recutita L.)

A protocol has been developed for the induction of somatic embryogenesis from flower explants of chamomile (Chamomilla recutita L.). The effects of several plant growth regulators [α-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA) and kinetin (Kin), alone or in combination] and the flower type (disk or ray flower) were investigated. Both types of flowers responded to the callus and shoot induction treatments, but formation of globular somatic embryos took place only on disk-flower-derived explants after 2–4 weeks of culture on a Murashige and Skoog (MS) medium supplemented either with 8.87 μm BA and 1.07 μm NAA or with 26.8 μm NAA and 11.5 μm Kin. However, fully developed, cotyledonary-stage somatic embryos could be induced only on the NAA/Kin medium, 10 weeks after culture initiation. Germination of the embryos and plant regeneration took place after subculture for 4–5 weeks onto medium of the same composition. Plantlets regenerated from embryos flowered in vitro on a MS medium supplemented with 8.87 μm BA and 1.07 μm NAA. The significance of the results with respect to chamomile micropropagation and the utilization of wild populations in breeding programs is discussed. Received: 6 April 1998 / Revision received: 12 October 1998 / Accepted: 28 October 1998     

Minimal growth conservation of potato microplants: silver thiosulfate reduces ethylene-induced growth abnormalities during prolonged storage in vitro

 Efficacy of silver thiosulfate (STS) in reducing ethylene-induced culture abnormalities during minimal growth conservation of microplants was studied in seven potato (Solanum tuberosum L.) genotypes. Different concentrations of STS (0, 1.5, 3.0, 4.5, 6.0, 7.5 and 9.0 μg ml^–1) were tested in minimal growth medium based on MS medium supplemented with 20 g l^–1 mannitol and 40 g l^–1 sucrose. STS improved the microplant growth and reduced the culture abnormalities during prolonged maintenance of potato shoot cultures in vitro. The beneficial effect of STS was most prominent for number of green leaves per microplant and leaf senescence. After 16 months of storage, desirable microplant growth was observed in cultures conserved in medium containing 6.0–9.0 μg ml^–1 STS. The profile of the peroxidase isozymes of conserved cultures did not show any apparent genetic variation due to the presence of STS in the conservation medium. Received: 2 September 1998 / Revision received: 20 November 1998 / Accepted: 12 December 1998     

Studies in the Physiology of the Onion Plant: VI. A SENSITIVE MORPHOLOGICAL TEST FOR BULBING AND ITS USE IN DETECTING BULB DEVELOPMENT IN STERILE CULTURE

Bulb development in the onion plant, which occurs in responseto the stimulus of long days, has two main morphological features:swelling of the base of the pseudostem and the formation ofscales from leaf initials produced at the stem apex. The swellingmay be expressed as the ‘bulbing ratio’, i.e. theratio of the maximum diameter at the base to the minimum atthe neck, but this is a somewhat insensitive measure wider adverselight conditions and no ratio can be specified at which bulbingis first apparent. In the absence of bulbing, leaf initialshave at all stages of development a blade longer than the leafsheath, giving a ‘leaf ratio’ of blade/sheath atall times greater than unity. In scale formation, when bulbdevelopment begins, the sheath of a small initial starts togrow sooner than in foliage leaf formation and blade growthis suppressed; the leaf ratio therefore falls rapidly belowunity and this is diagnostic of bulbing. Characteristic graphsof leaf ratio plotted against leaf number from the apex demonstratethe stage of bulbing reached and a fall in leaf ratio from nearthe apex in such a graph can indicate the beginning of bulbingbefore any actual scales (with ratios less than unity) havebeen formed. In normally grown plants, differentiation intofoliage leaves or scales appears to take place when the initialsreach about 1 mm in length. When the incidence of bulbing within a treatment is very variable,difficulties arise in averaging the leaf ratio curves for thedifferent plants. Mean number of scales per plant, or mean minimumleaf ratio, may then be used as measures of bulbing. Heavy shading in winter was found to delay but not prevent thebulbing of those plants which survived. ‘Buds’, consisting of a portion of stem with theapex surrounded by leaf initials, but no scales, were dissectedout from onion sets and cultured aseptically in tubes. Limitedgrowth was obtained and rooting occurred in some cases. In thepresence of sucrose, swelling of leaf sheaths and reductionof the leaf ratios took place; these responses increased withsucrose concentration. Glucose similarly stimulated bulbingbut high osmotic pressure caused by mannitol or mineral nutrientshad no effect. Rooting appeared to reduce the degree of bulbing,possibly by competing for carbohydrate supply. The bulb developmentin these sterile cultures was most unusual in occurring in shortphotoperiods and the possibility of a residue of ‘bulbinghormone’ being stored in sets is discussed.     

Differentiation in Lilium Bulbscales Grown in vitro. Effect of Various Cultural Conditions

Tissue cultures of Lilium auratum Lindl. and L. speciosum Thunb., which were derived from bulbscales, all appeared to differentiate organs. The effect of cultural conditions on the differentiation of bulblets and roots was examined. The best material for bulblet formation was bulbscales of intact or in vitro produced bulblets. The optimum temperature was 20°C and optimum pH was 6. Effect of irradiance on organ formation was not obvious but leaf emergence was stimulated. Higher kinetin concentrations stimulate the formation of numerous bulbscalcs. High NAA concentrations induce roots. On the other hand kinetin inhibits the NAA effect on root formation. A high sucrose concentration stimulated organ formation, but the number of bulblets was at a constant level in the medium containing between 10 and 90 g/l of sucrose. The formation of bulblets and their growth were stimulated at increasing strength of Murashige-Skoog's (MS) medium, but the length of roots was inhibited. Inter action of strength of MS medium and sucrose concentration was examined. High concentration of both components stimulated bulb lei growth, but the second strength of MS medium containing 90 or 120 g/l sucrose stimulated callus induction and inhibited the growth of bulblets. Maximum growth took 100 days for bulblets and about 50 days for roots. The change of fresh weight/dry weight ratio during differentiation is also discussed.     

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique

 Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10–50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized. Received: 13 September 1999 / Revision received: 2 March 2000 / Accepted: 16 March 2000     

Cryopreservation of Panax ginseng cells

The impacts of cryoprotectants (CP) and cell status during the growth cycle on Panax ginseng cell viability during cryopreservation were investigated. The ginseng cells used had a 5–7 times proliferation rate (compared with inoculum) in 2–3 weeks and were subcultured at 2- and 4-week intervals in liquid and on solid media, respectively. After testing various CP solutions of glycerol, dimethylsulphoxide, ethylene glycol and sucrose, a combination of 10% (v/v) glycerol and 4% (w/v) sucrose was selected for its least cytotoxicity and highest cell viability after thawing. With this CP solution, cells throughout the growth cycle exhibited a ‘U’-shaped fluctuation of post-thaw cell viability. The highest viability (86.5%) occurred during the lag phase from cells already maintained in suspension culture and then in the late exponential phase (61.7%); the lowest level of 15.4% was in the mid-exponential phase. Callus freshly transferred to liquid medium showed a less obvious fluctuation pattern. The recovered cells were brown-to-reddish at first and gradually returned to a light yellow colour after several subcultures. Received: 1 October 1998 / Revision received: 6 January 2000 / Accepted: 11 January 2000     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000     

Use of recombinant inbred lines (RILs) to identify, locate and map major genes and quantitative trait loci involved with in vitro regeneration ability in Arabidopsis thaliana

The Landsberg erecta× Columbia recombinant inbred lines (RILs) of Arabidopsis have been used in order to identify and localize chromosome regions involved in the genetic control of the in vitro regeneration ability. Callus morphology (CM) and shoot regeneration (SR) traits have been considered for both leaf and root explants. The MAPMAKER analysis of leaf culture data has revealed at least one chromosome region involved with CM and several with SR, the 29–30 region of chromosome 1 being common for the two traits. Root explants did not segregate for CM but several QTLs have been detected for SR. The chromosome regions involved with leaf culture regeneration seem to be different from those of root cultures, although the regeneration of abnormal shoots in leaf explants share two chromosome regions with the regeneration of normal shoots in root cultures. Received: 19 April 2000 / Accepted: 12 May 2000     

In vitro sucrose concentration affects growth and acclimatization of <Emphasis Type="Italic">Alocasia amazonica</Emphasis> plantlets

Plantlets of Alocasia amazonica were regenerated on the MS medium supplemented with different concentrations (0–9%) of sucrose. An absence of sucrose in the growth medium induced generation of leaves, however, it decreased multiplication. On contrary, sucrose supply of 6% or 9% enhanced multiplication but hampered photoautotrophic growth (generation of leaves). Increasing sucrose supply also increased sugars and starch content and number of stomata and decreased water potential and size of stomata during in vitro growth period. During ex vitro acclimatization, shoot length, root length, leaf number and root number of Alocasia plantlets grown with 3% sucrose, were found to be better among the other studied sucrose concentrations. Under ex vitro acclimatization, number of stomata, contents of various carbohydrates in the leaves were increased but size of stomata decreased with increasing sucrose supply during in vitro growth period. Moreover, water potential of leaves of plantlets, which have been grown with a sucrose concentration other than 3%, was decreased. During in vitro growth, net CO₂ assimilation rate (P_N), transpiration (E), stomatal conductance (g_s) and variable fluorescence to maximum fluorescence ratio (Fv/Fm) were unaffected, however, during acclimatization these were changed and maximum P_N, E, and g_s were observed in the plantlets micropropagated with 3% sucrose. Fv/Fm was decreased severely in the plantlets micropropagated with 6% sucrose during acclimatization. Thus a sucrose concentration of 3% in the medium is appeared to be better among studied concentrations for both in vitro growth and ex vitro acclimatization of A. amazonica plantlets.     

In vitro propagation of Herreria salsaparilha Martius (Herreriaceae) as affected by different sealing materials and gaseous exchanges

The effects of sealing material on the gaseous composition inside flasks containing Herreria salsaparilha in vitro cultures, and on axillary shoot propagation of this species were evaluated. Axillary shoots (1.5 cm in size) were cultured in 200 ml flasks containing 45 ml MS medium supplemented with 30 g l⁻¹sucrose, 100 mg l⁻¹ myo-inositol, MS vitamins and 5 g l⁻¹ agar. Flasks were sealed with polypropylene rigid closure (C), polypropylene rigid closure with a filter (CF), or single- or double-layer polyvinylchloride films (PVC1 and PVC2, respectively). Cultures were maintained at 26 ± 1°C, and a 16/8 h light/dark photoperiod with 35 μmol m⁻² s⁻¹ irradiance. After 60 days, water loss (%), internal O₂ concentration (%), plant height (cm), leaf and root fresh and dry weights (g), and root number and length (cm) were evaluated. Using an open system infrared gas analyzer, the photosynthetic and respiratory rates of in vitro H. salsaparilha cultures were also estimated. The sealing material influenced water loss in flasks, with CF closure showing higher percentage of water loss. O₂ concentration was significantly higher in PVC-sealed flasks than in other treatments, leading to the formation of a small dome on top of the flask. Despite the high oxygen accumulation, shoot development was not affected.     

An efficient system for the production of clonal plantlets of the medicinally important aromatic plant: Salvia africana-lutea L.

An in vitro cultivation protocol was developed for S. africana-lutea a species threatened by over collection due to its importance as an aromatic medicinal plant in the Western Cape of South Africa. Adventitious shoot induction was most successful using hypocotyls as explants for propagation on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with 4.4 μM BA only; 2.7 μM NAA and 4.4 μM BA; or 2.9 μM IAA and 9.3 μM kinetin respectively. For continuous subculture, IAA and BA (μM) at a ratio of 2.9:4.4 or 2.9:8.9 had the best regeneration potential producing approximately three plantlets per nodal explant. Plantlets had 4–5 nodes that could be utilized for the following subculture phase to induce axillary shoots. The tissue culture of S. africana-lutea not only favoured rapid multiplication but was also characterized by seasonal in vitro flowering that was in synchrony with that of plants growing in the wild. This propagation regime has the capacity for producing 2000–3000 plants from one shoot after 3 four-week long subculture cycles, making it highly attractive for implementation as an in vitro conservation strategy. The micropropagated plants were easily acclimatized (88%) within a month after rooting in vitro and planted ex vitro in a sand:soil:peat moss:vermiculite (1:1:1:1; v/v) mixture.     

Enhanced production of saikosaponins by root culture of Bupleurum falcatum L. using two-step control of sugar concentration

 The effect of sugar concentration on the production of saikosaponins was investigated using a root culture of Bupleurum falcatum L. The formation of the lateral roots, which were induced in the presence of indolebutyric acid, was suppressed as the sugar concentration was increased. After the lateral root tips had emerged from the inoculated roots, however, high concentrations of sugar showed no inhibitory effect on the development of the lateral roots. A two-step culture, with 1% sucrose at the beginning of the culture and addition of 6% sucrose at 14 days, when lateral roots have emerged, greatly improved the productivity, affording 0.8 g/l of saikosaponin-a and -d. Received: 28 October 1999 / Revision received: 19 April 2000 / Accepted: 27 April 2000     

Direct rhizogenesis and establishment of fast growing normal root organ culture of Withania somnifera Dunal

Direct rooting from leaf explants of Withania somnifera was achieved on half strength Murashige and Skoog’s medium supplemented with 15 g l⁻¹ sucrose, and different concentrations of growth regulators. Basal medium supplemented with 2.85 μM indoleacetic acid and 9.85 μM indolebutyric acid achieved maximum number of roots with 100% response. The roots were cultured on MS liquid medium for the establishment of root-organ culture with the same plant growth regulators and incubated on an orbital shaker at 80 rpm at 25 ± 2 °C. A root biomass of 6.15 ± 0.17 g was obtained after 5 weeks. When 1 g roots were inoculated to 2.5 l bubble column reactor, 47 g roots were obtained after 6 weeks. The concentration of alkaloids was increased as compared to field grown roots. The maximum concentration of withanolides (10 mg g⁻¹ dry weight) was obtained in the bioreactor.     

A protocol for in vitro mass propagation of asiatic hybrids of lily through liquid stationary culture

Summary A simple, rapid and cost-effective in vitro scheme has been proposed for mass propagating two cultivars of Asiatic lily hybrids. An average of seven bulblets was formed after 17 d when 1×1 cm² bulb scale segments (explants) were cultured on Murashige and Skoog (MS) medium with 3% sucrose and 0.5 μM α-naphthaleneacetic acid (NAA). On MS medium containing 0.5 μM NAA and 6 or 9% sucrose, depending on the cultivar, large numbers of bulblets of increased size (3.5–5.0 cm in circumference) were formed under a 16/8 h photoperiod. A continuous system of mass propagation of bulblets was achieved through in vitro scale formation (secondary explants) on MS medium supplemented with 23 μM kinetin and 0.5 μM NAA, as well as scale proliferation on MS basal liquid stationary medium. Upon transplantation all bulblets sprouted, of which 40% flowered in the first season. Under ideal conditions, ca. 9.68×10⁵ bulblets can be produced from a single scale segment in 1 yr by following the systematic propagation steps proposed here.