GROWTH IN VITRO OF TISSUES ISOLATED FROM NORMAL STEMS AND INSECT GALLS

Pelet , F., A. C. Hildebrandt , A. J. Riker, and F. Skoog . (U. Wisconsin, Madison.) Growth in vitro of tissues isolated from normal stems and insect galls . Amer. Jour. Bot. 47(3) : 186—195. Illus. 1960.–In a preliminary analysis of the nature of gall formation induced by insects, a comparative study has been made of the in vitro growth and nutrition of plant tissues derived from insect galls and from normal plants. Grape, elm, poplar, and willow tissues were grown on a standard medium, modified White's nutrient medium, with coconut milk and/or various growth factors added. Satisfactory growth was obtained over a temperature range from 16° to 36°C. but was generally optimal at 28°—32°C. The optimum pH was generally 4.0—4.5, but a pH of 6.0 or 7.0 gave better growth when the medium contained 2,4-dichlorophenoxyacetic acid. Detailed nutritional studies were limited to grape tissue. Excised stems and excised galls induced by Phylloxera vastatrix Planch, were grown on the basal medium with vitamins and supplemented with naphthaleneacetic acid, indoleacetic acid, kinetin, casein hydrolysate, yeast extract, adenine and a few amino acids added in various combinations. Growth (fresh weight) was measured after a 6-week growth period. When these substances were added singly the optimal concentrations and the quality of growth of stem explants were as follows: with adenine (40 mg./l.) or kinetin (1 mg./l.), growth poor; with NAA (1 mg./l.) or IAA (2 mg./l.), growth fair; and with the only concentration of a powdered casein hydrolysate (3 g./l.), growth good. Gall explants responded more readily to kinetin or adenine but did not form callus in the presence of casein hydrolysate alone. Stem tissues formed both roots and callus, whereas gall tissues formed only callus. The same substances were tested in various combinations. NAA and kinetin provided for moderate, continuous growth, and excellent growth if casein hydrolysate and adenine also were added to the medium. The NAA requirements were markedly reduced in the grape tissues which had been subcultured for 1 or 4 years on coconut milk medium. Friable tissue types were inhibited by the adenine and casein hydrolysate combinations. They grew through 1 passage only on basal medium and then died if not supplied with NAA and kinetin. Firm tissues responded favorably, although irregularly, to casein hydrolysate and adenine. It was concluded that although nutrient requirements varied with tissues derived from insect galls and from normal plants, they also varied with the time of cultivation in vitro. The induction of galls by Phylloxera was not a permanent change in growth factor requirements comparable to that conferred by the crown gall bacteria. In attempts to grow the insect in sterile culture in vitro 5 successive generations of phylloxera were reared on callus tissue.     

TISSUE CULTURE OF CALLUS FROM SEEDLING AND ADULT STAGES OF HEDERA HELIX

Callus from stem pieces of the juvenile (seedling) and adult stages of Hedera helix L. were grown for up to 17 passages on a medium containing mineral salts, vitamins, sucrose, and coconut water and for fewer passages on media which lacked coconut water. Seedling callus differed in its growth in culture from adult. It was less stable than the adult; i.e., it developed vigorous variants earlier and more freely than the adult callus. The tendency of seedling callus to adapt to unfavorable media was greater. On low salt media root formation by callus was infrequent. Adult calluses formed roots more freely on high salt media than seedling calluses did.     

AUXIN,CYTOKININ AND GROWTH OF EXCISED ROOTS OF BRYOPHYLLUM CALYCINUM

Excised roots 1 cm long of Bryophyllum calycinum required exogenous auxin (α-naphthaleneacetic acid) for growth in still culture in a medium of sucrose, mineral salts and vitamins. Little or no growth occurred with the addition of cytokinin (6-benzylaminopurine) alone to the basal medium. Best growth was obtained with combinations of 1 μg of auxin and 1 or 2 μg of cytokinin in 50 ml of medium. Larger amounts of the two phytohormones resulted in reduced growth.     

Factors affecting callus and shoot formation from in vitro cultures of Lens culinaris Medik.

The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these calli. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg l^–1 of BA and 0.186 mg l^–1 NAA+2.25 mg l^–1 BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.     

In vitro induction of multiple shoots and plant regeneration from shoot tips of mung bean (Vigna radiata (L.) Wilczek)

Conditions for plant regeneration from excised shoot tips of Vigna radiata were studied. Complete plants were regenerated directly without an intervening callus phase from shoot tips on basal medium (MS salts+B₅vitamins). Regeneration frequency varied with genotype, explant size and growth regulator combinations in the medium. Addition of cytokinins induced a variable amount of callus at the base of the shoot tip, followed by multiple shoot formation. Benzyladenine (BA), kinetin and zeatin at 5×10^-6 M each induced multiple shoots in 100% of the explants but the highest number of regenerants per explant (9) was produced with BA. The efficacy of BA for shoot multiplication was not improved when it was supplemented with naphthaleneacetic acid (NAA) or indoleacetic acid (IAA). NAA or adenine sulphate, when applied alone, induced complete plantlets. The growth regulator requirement of explants for the induction of multiple shoots varied with explant size. The shoot tip explants maintained proliferation ability on subculture. None of the treatments was effective in inducing shoot bud differentiation from callus. Regenerated shoots were rooted on MS basal medium and MS supplemented with either IAA or indolebutyric acid. The rooted plants were transferred to the field; 60% subsequently survived and grew.Abbreviations BM basal medium [MS (Murashige & Skoog 1962) salts+B₅ (Gamborg et al. 1968) vitamins] - BA 6-benzyladenine - AdS adenine sulphate - IAA indole-3-acetic acid - NAA-1 naphthaleneacetic acid - IBA indolebutyric acid     

Embryogenic callus formation,growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda Giganteus' as affected by proline

The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22.6 μM 2,4-dichlorophenoxyacetic acid. Shoot apices and leaves from in vitro-propagated shoots, and immature inflorescences from greenhouse-grown plants were used as explants for callus induction and formation. Suspension cultures initiated from embryogenic callus of immature inflorescences were used to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates. An optimum proline concentration for plant regeneration was found at 12.5 mM. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration in two-year old cultures of Zea diploperennis

Immature embryos and immature leaf tissues were used to establish embryogenic cultures of Zea diploperennis. Callus was induced on media containing MS salts and vitamins, sucrose (2% for leaves, 6% for embryos), 5% coconut milk and 1–6 mg/l 2, 4-D. Embryogenic callus was maintained by subculturing on media containing MS salts and vitamins, 2% sucrose, 500 mg/l casein hydrolysate and 1 mg/l 2,4-D. Regeneration occurred when the 2,4-D level was reduced to 0.25 mg/l. Kinetin added at 0.25 mg/l further stimulated regeneration. Root tip squashes on 10 plants regenerated after 2 years in culture indicated a normal 2n=20 chromosome number.     

Studies on some factors affecting solasodine contents in tissue cultures of Solanum nigrum

Tissue cultures of Solanum nigrum L. were initiated from leaf explants on a solid medium containing inorganic salts [Murashige and Skoog (1962), Physiol. Plant. 15: 473–497], vitamins [Gamborg et al. (1968) Exp. Cell Res. 50:151–158], 3% sucrose and combinations of indoleacetic acid and benzyladenine. Solasodine content was determined in differentiated and undifferentiated (callus) tissues by a colorimetric technique and thin layer chromatography. Indoleacetic acid and sucrose in the medium markedly stimulated the production of solasodine in the tissue cultures. In the cultures grown in darkness the differentiated tissues produced significantly more (anywhere from 1.5 to 10 times) solasodine than the callus in several media. When sucrose concentration was increased to 4, 6 and 10% level in the medium which contained 10 μ M benzyladenine as the sole growth regulator, a significant increase of solasodine production in cultures was found.     

The Effect of Various Media on the Growth Responses of Different Clones of Carrot Explants

The induction of growth in otherwise quiescent tissue explantedfrom carrot root has been investigated with reference to theeffects of different kinds of growth-promoting substances addedas supplements to a basal medium, singly and in combination.The effects of these media upon different clones of carrot explantsare described. The idiosyncrasies of different clones of explantswere detected by their responses measured by the incidence ofcell division, the extent of cell enlargement, and by theirnucleic acid content. The basal medium which contains salts,sugar, and vitamins supported only a minimal amount of growth;the basal medium supplemented with casein hydrolysate and coconutmilk (10 per cent by volume) supported the highest level ofgrowth obtained in any of the treatments tried. The active componentsof the coconut milk (AF_cm) when refined required the furtherparticipation of either indole-3yl-acetic acid (IAA) or inositol,and were further stimulated by casein hydrolysate (CH). Thusthe over-all stimulus of the coconut milk comprised two parts—nowrecognized as growth-promoting systems I and II, respectively.The effects of System I were mediated by appropriate combinationsof inositol and the corresponding active growth-promoting factors(AF₁) which were, in turn, represented by a purified factorpreviously isolated from Aesculus (AF₂). System I induced bothcell division and cell enlargement in balance, whereas SystemII stimulated internal cell division more than cell enlargement.The effects of System II were mediated by appropriate combinationsof IAA and active growth-promoting factors (AF₂), which wererepresented by the substance zeatin. The maximum growth of anygiven clone of carrot explants isolated from a given carrotroot was only supported by exogenous requirements, over andabove a basal nutrient medium, which meet its specific endogenouslimitations. The paper shows how these limitations may be diagnosed,and discusses the over-all growth stimulus due to coconut milkin terms of the partial responses elicited by the known componentsof Systems I and II.     

In vitro differentiation of plantlets from embryonic explants of lodgepole pine (Pinus contorta Dougl. ex Loud.)

A protocol is described for plantlet formation in juvenile tissues of Pinus contorta. Shoots were induced on embryonic, cotyledonary and hypocotyl explants cultured on a defined medium supplemented with cytokinin. The concentration of salts, vitamins and cytokinin (benzylamino purine) in the medium, as well as different temperature regimes, strongly influenced the frequency of bud formation. Differentiation of shoot primordia and their subsequent development was also markedly affected by cytokinin exposure times. Bud development and elongation were enhanced by elimination of the phytohormone, reducing the strength of mineral salts, vitamins and sucrose in the medium, as well as by the inclusion of charcoal. Rooting was induced by treating the shoots with a sterilized rooting powder containing indole-butyric acid and culturing them in agar-solidified medium containing reduced mineral salts, vitamins, sucrose and charcoal. The number of chromosomes and their structure were found to be normal in the regenerated plantlets.     

Somatic embryogenesis and plant regeneration in embryo cultures of Euterpe edulis mart. (palmae)

The induction of somatic embryogenesis in embryo cultures of Euterpe edulis is described. The basal medium was composed of LS salts and Morel & Wetmore vitamins. Activated charcoal was added to prevent explant oxidation. 2,4-D higher than 50 mg/l was necessary for inducing embryogenesis which occurs 45–180 days after the start of cultures. Embryos arise directly from surface proliferating tissues on the matrix structure , without callus formation. The transfer of tissues with embryo clusters to medium with NAA plus 2iP, or without growth regulators, induces embryo development into plantlets.     

Adventitious shoot regeneration of scotch spearmint (menthaxgracilis Sole)

Summary The effect of different cytokinins on in vitro adventitious shoot regeneration from internodal explants of Menthaxgracilis Sole (scoth spearmint) was investigated. Murashige and Skoog (MS) medium containing 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% (w/v) sucrose, 10% (v/v) coconut water and supplemented with 4.5 μM thidiazuron (TDZ) was effective in inducing adventitious shoot formation from callus. The greatest percentage of explants with shoots (85%) with the highest mean number of shoots per explant (29) was obtained with explants from the 1st and the 2nd internodes from 2-wk-old stock plants growing on a medium containing MS basal salts, 2% sucrose, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, at TDZ 4.5 μM and 10% (v/v) coconut water and solidified with 0.2% (w/v) phytagel. The regenerated shoots rooted on a medium containing MS basal salts, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% sucrose, and 0.054 μM naphthalene acetic acid (NAA). Micropropagated plantlets were transplanted into soil and acclimated to greenhouse conditions. This is the first report describing adventitious shoot regeneration of scotch spearmint.     

Genetic transformation ofEustoma grandiflorum byAgrobacterium tumefaciens

Callus cultures were initiated from micropropagated Artemisia absinthium plantlets on MS basal medium supplemented with different concentrations of BA, Kn, NAA, IAA and 2,4-d in combination or singly. Supplementing the medium with low doses of both BA in combination with NAA, and Kn in combination with NAA enhanced the growth rate of callus cultures. However, cultures grew slowly following the second subculture and the majority turned brown and died within the next month. Initiation of root and shoot primordia occured directly from leaf explants cultured on 1.81 M 2,4-d, while adventitious shoot formation from callus was observed occasionally when BA was added to the medium in combination with IAA. Furthermore, medium containing 2.22 M BA and 2.69 M NAA stimulated both callus growth and organogenesis on some callus cultures derived from leaves and stems of young stock material. The best results were obtained with leaf explants. Cytological analysis of root meristems revealed that all regenerants were diploid (2n=18), as expected.Abbreviations MS Murashige & Skoog's salts and vitamins (1962) - BA 6-benzyladenine - NAA alphanaphthaleneacetic acid - Kn Kinetin (6-furfurylaminopurine) - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - FW fresh weight - Bi biomass increase     

Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and Gracilaria perplexa (Gracilariales, Rhodophyta)

The effects of auxins and cytokinin on callus formation, growth and regeneration of Gracilaria tenuistipitata Chang et Xia and G. perplexa Byrne et Zuccarello (Gracilariales, Rhodophyta) are reported. Plant growth regulators (PGR) in concentrations ranging from 0.1 to 100.0 μmol of indole‐3‐acetic acid, 2,4‐dichlorophenoxyacetic acid (2,4‐D), and kinetin (K) were added to the ASP 12‐NTA solid medium (0.7% agar), and apical and intercalary segments (5 mm long) were inoculated as initial explants. K stimulated growth rates of intercalary segments of G. tenuistipitata in a linear relation, and 2,4‐D (1.0 μmol) and K (10.0 μmol) stimulated growth rates of apical and intercalary segments of G. perplexa, respectively. The simultaneous formation of apical, basal, and intermediate calluses is reported for the first time in axenic tissue cultures of red algae. With intercalary segments of G. tenuistipitata, basal callus induction rates were higher than those of apical and intermediate calluses in the majority of treatments, and auxins had stimulatory effects on the formation of all callus types. In apical segments of G. perplexa, intermediate callus formation was stimulated only by treatment with 1.0 μmol of K, while apical callus formation was stimulated by indole‐3‐acetic acid (1.0–10.0 μmol), 2,4‐D (10.0–100.0 μmol), or K (0.1 μmol). Intercalary segments of G. perplexa developed only intermediate calluses, and the majority of treatments with PGR stimulated higher rates than those presented by apical segments. Potential for regeneration (development of adventitious plantlets originated from callus cells) was higher in apical calluses than in basal and intermediate calluses developed in intercalary segments of G. tenuistipitata. Moreover, auxins and cytokinin were essential to the induction of regeneration in intermediate calluses, while specific concentrations stimulated regeneration from basal and apical calluses. Plant regeneration in G. perplexa was observed only after transferring calluses from solid to liquid medium, and the majority of treatments with PGR had stimulatory effects. Regenerating plants of G. perplexa developed tetrasporangia, and released tetraspores giving rise to adult gametophytes. Our results indicate that auxins and cytokinin have a regulatory role in the growth and morphogenesis in G. tenuistipitata and G. perplexa, and diversity of responses presented by both species is related to specific developmental systems.     

植物生长调节剂对南方红豆杉愈伤组织培养和紫杉醇合成的影响

通过不同种类和水平植物生长调节剂对南方红豆杉(Taxus chinensisvar.mairei)愈伤组织诱导、生长和紫杉醇合成能力影响的研究发现:诱导培养初期,以无植物生长调节剂的MS为基本培养基,在附加不同植物生长调节剂组合作用下愈伤组织产生的时间和生长、在相同植物生长调节剂组合作用下不同外植体愈伤组织的产生时间和生长均表现出较显著差异,2,4-D/NAA高于0.4时,不利于南方红豆杉愈伤组织的诱导。转换到附加不同植物生长调节剂组合的B5培养基上后,随培养继代次数的增加,生长差异逐渐缩小,直至不显著,表明参考不同文献报道最优配方所设计的各植物生长调节剂组合对南方红豆杉愈伤组织的生长均较适宜,有利南方红豆杉愈伤组织生长的植物生长调节剂优化组合没有唯一性。但不同调节剂组合作用下的同源愈伤组织中、相同调节剂作用下不同源愈伤组织中紫杉醇含量均存在着极显著差异,适当水平(2 mg/L)的2,4-D单用,或与适当水平的KT、6-BA、KT GA配合使用,对南方红豆杉愈伤组织紫杉醇的合成较有利,NAA则不太有利,幼茎和叶愈伤组织产紫杉醇的水平较其它愈伤组织为高。     

Preliminary observations on organogenesis inTaraxacum officinale tissue cultures

Summary Tissue cultures ofTaraxacum officinale have been isolated from the secondary thickened root. Callus development and leaf and root formation occur on a basal medium supplemented with coconut milk and IAA or NAA, and the addition of kinetin to these media enhances callus growth and organogenesis. Cultures grown on the basal medium with coconut milk and 2,4-D show only callus growth, but organogenesis is induced by the substitution of IAA for 2,4-D. In the 2,4-D grown callus a layer of secondary meristematic tissue is present and organogenesis apparently occurs from localized regions of this tissue which have undergone de-differentiation to the primary meristematic condition.     

Efficient plant regeneration of native spearmint (Mentha spicata L.)

Summary Protocols and media constituents for efficient in vitro plant regeneration of Native Spearmint (Mentha spicata L. cultivar ‘Native Spearmint’) have been defined. Adventitious shoots were initiated either directly from morphogenetically competent cells of explants or primary callus. Leaf explants from at least 2-mo.-old in vitro-maintained shoots exhibited the greatest morphogenetic capacity. Explants derived from basal portions of leaves at the bottom of the shoot were most responsive, with up to a 100% regeneration frequency and greater than nine shoots per explant. Highest frequency of meristemoids and morphogenetic callus were initiated from explants cultured onto a basal medium containing Murashige and Skoog (MS) salts, supplemented with 4 mg thidiazuron (TDZ) per L and 25% (vol/vol) coconut water (CW) for 10 to 14 d in darkness. Bud and shoot development required removal of both TDZ and CW from the medium. Shoot propagules were transferred to basal medium supplemented with 0.01 mg α-naphthaleneacetic acid (NAA) per L and grown under low light for about 2 wk to facilitate shoot elongation. Individual shoots about 1 cm tall were dissected and retransferred onto the same medium. Root initiation began within 4 to 6 d and a functional root system developed within 2 to 3 wk. These plantlets were transferred to soil and acclimated successfully for growth and development in a greenhouse. This is the first report of an efficient regeneration system for Native Spearmint based on adventitious organogenesis.     

Differential effects of sucrose, abscisic Acid, and benzyladenine on shoot growth and callus formation in the abscission zone of excised citrus buds

The omission of sucrose from the basal medium stimulated callus formation in bud explants of Citrus sinensis (L.) Osbeck. Moreover, it increased the abscisic acid-induced callus proliferation reported earlier in the presence of 5% sucrose (Altman and Goren, Physiol. Plant. 32: 55, 1974). The inhibition of callus formation by the addition of sucrose was not due to the high osmotic potential of the medium. Benzyladenine induced callus formation slightly, in all sucrose concentrations up to 5%. The high level of sucrose was required, however, for the growth of shoots from buds cultured on both basal and benzyladenine-containing media.     

Actinidia deliciosa C.F. Liang in vitro

In vitro growth of Actinidia deliciosa C.F. Liang, cv Hayward and changes in mineral composition of the medium and in the different parts of the explants (callus, stem and leaves), were analyzed after 0, 15, 30, 45 and 60 days of culturing in each of three successive 60 days subcultures.Fresh (FW) and dry weight (DW) of the explants increased mainly during the first 30 days of culturing, with a predominant increase of FW in leaves and an equal distribution in DW in callus and leaves. Stem FW and DW changes were lower than those observed with callus and leaves. As FW and DW of the explants increased the FW and DW of the medium decreased.The presence of the explants induced a large decrease of medium pH during the first 15 days of culturing followed by a return to the initial level at the end of the culturing.The initial P content of the MS medium was insufficient for the long term culturing, as after 30 days of culturing almost all (94.5%) the P present in the medium was absorbed by the explants and evenly distributed in their different parts. During the first month, 85% of the initial N was absorbed. At the end of the culture only 2% of the initial P and 5% of N remained in the medium. These two elements were equally distributed in callus and leaves during the first month of culturing, while in the last 30 days they increased only in the callus.MS medium initial concentrations of K, Mg, Ca, Fe, Zn, Mn and Cu were sufficient for 60 days explants growth. Almost all these elements were absorbed during the first 30 days of culturing. Their distribution in the different parts of the explant was uneven throughout the culture period. Callus tissue was the main site for accumulation of all these mineral elements.     

Overcoming phenolic accumulation during callus induction and in vitro organogenesis in water chestnut (Trapa japonica Flerov)

Summary Procedures for callus induction and subsequent organogenesis in the aquatic plant, water chestnut (Trapa japonica Flerov), were established. Phenolics exuded from explants at the callus-induction stage adversely affect callus growth. For cotyledonary node-derived callus cultured in Murashige and Skoog (MS) medium (full, half or quarter strength) containing 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with benzyladenine (BA), the accumulation of phenolics was reduced and callus induction increased by the addition of 10.8 μM phloroglucinol (PG) to the medium. Ascorbic acid was also effective in reducing phenolic accumulation, but less effective for callus induction than PG. Half-strength MS medium supplemented with 2.7 μM 2,4-D, 108.0 μM casein hydrolyzate, and 10.8 μM PG supported maximum callus induction. Plant organogenesis was increased by addition of vitamins (0.27 μM biotin and 2.7 μM folic acid) to half-strength MS medium supplemented with 0.27 μM BA. Many shoots developed from the regenerated nodal shoot explants in liquid half-strength MS salts medium supplemented with 1.08 μM BA and 0.27 μM naphthaleneacetic acid. Individual shoots were excised and cultured in liquid half-strength MS medium supplemented with 5.4 μM IBA and rooted plantlets (108) were transferred and acclimatized in plastic pots. After 3 wk, the plantlets were transplanted in a water chestnut field and the survival rate was 100%.