Growth Hormones and Propagation of Cymbidium in vitro

Protocorms of Cymbidium (Orchidaceae) were grown on solid or liquid medium with macro-nutrients according to Wimber (van Raalte 1967) and iron, micro-nutrients and vitamins according to Nitsch (1968) the medium also contained 2% sucrose. The effects of 1) the auxins; indol-3yl-acetic acid (IAA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D); 2) the cytokinins; 6-furfurylaminopurine (kinetin) and benzyladenine (BA) and 3) the gibberellin; gibberellic acid (GA) were examined alone or in combinations. IAA had no effect alone. NAA resulted in optimal fresh weight at 10 μM and the protocorms were vigorous, but lighter green than usual. 2,4-D caused a high weight increase at 1 μM, but the protocorms were abnormal. Higher concentrations of NAA and 2,4-D inhibited chlorophyll synthesis. On solid medium kinetin (100 μM) induced a growth of many small shoots, but had no effect on the fresh weight. In liquid medium, kinetin promoted a callus formation and fresh weight increase. BA had effects similar to kinetin, but at lower concentrations. GA alone promoted shoot and leaf growth. Combinations of kinetin and NAA resulted in a maximal fresh weight increase at kinetin concentrations one tenth of the NAA concentrations. The optimal growth and the best development occurred at 10 μM NAA and 1 μM kinetin. NAA and kinetin together could limit the shoot and leaf growth induced by GA.     

Induction of apogamy inEquisetum arvense

InEquisetum arvense, apogamous sporophytes were produced on medium containing 5×10^?6–5×10^?8 g/ml kinetin. NAA, IAA, GA₃, glucose and saccharose were ineffective for the induction of apogamy. On medium containing 5×10^?7–5×10^?8 g/ml kinetin, the gametophytes passed into sporophytic structures directly. On medium containing 5×10^?6 g/ml kinetin, some gametophytes passed into sporophytic structures directly, and others became a callus-like cell mass from which an apogamoun shoot arose. The results of the morphological observations on them were reported and compared with the sexually produced sporophyes. The apogamous sporophytes induced by 5×10^?7 g/ml kinetin were haploid in their nuclear phase and some of those induced by 5×10^?6 g/ml kinetin had a tendency to become diploid.     

THE INFLUENCE OF CULTURAL CONDITIONS ON THE INDUCTION OF APOGAMY IN PTERIDIUM GAMETOPHYTES

Apogamous sporophytes formed on Pteridium gametophytes in response to concentrations of certain sugars which supported gametophytic growth. High osmotic concentration of the medium inhibited apogamy, while variations in the basic medium were not stimulatory. Agar, autoclaving, the ammonium ion, and dry media were not required for apogamy. Renewing the medium during an experiment enhanced the apogamous response. Changing the medium at set intervals facilitated the separation of apogamous plant development into gametophytic, initiative, and developmental phases, thus enabling testing of various factors at each of these stages. Apogamy was light-initiated, while the actual development of apogamous sporophytes was caused by light, succinic acid or sugar.     

Response of Pusa Seedless Grape to 4-CPA, Kinetin, Uracil and GA

Response of Pusa Seedless, a cultivar of Vitis vinifera to 4-chlorophenoxyacetic acid (4-CPA), kinetin, uracil and gibberellic acid (GA) separately and in combinations was tested. Dipping the clusters in GA at 50–100 mg/1 at 2–3 days after full bloom significantly increased the bunch and berry weight of Pusa Seedless Grape. 4-CPA and uracil treatments increased the bunch and berry weight to some extent but not as effectively as GA treatments and kinetin had no effect. GA proved to be the best in increasing the berry weight of this cultivar. A synergistic effect was observed when 4-CPA was combined with GA at 50 mg/1 but combinations of 4-CPA with higher concentrations of GA did not show such synergistic effect. In general, GA alone and especially in combination with 4-CPA lowered the quality of the berries. Uracil on the other hand, improved the quality when applied alone and in combination with GA.     

EXPERIMENTAL CONTROL OF THE SHOOT SYSTEM IN SPORELINGS OF PTERIDIUM AQUILINUM

The shoot system of Pteridium develops from a simple, upright sporeling type through a rhizomatous leaf-bearing form, into a complex system with rapidly growing, leafless long shoots and slowly growing, leafy lateral branches or short shoots. Transitional stages axe available in sporeling material and ontogeny may be controlled by specific carbohydrate levels. At concentrations of sucrose below 2% a rhizomatous sporeling plant may be maintained indefinitely in the leaf-bearing state. At 2–8% sucrose, the short shoot habit becomes established, and at 4–8% it may be reliably stabilized. If concentrations up to 5 mg/liter of kinetin are added to cultures of intact plants or to excised short and long shoot apices of plants, the long shoots expand slowly into determined (no further growth possible even on transfer back to basal medium) club-shaped tips. The short shoot apices, by contrast, quickly proliferate into callus-like masses and will re-form organized meristems over the entire lobulate surface on transfer to medium lacking kinetin. The morphogenetic significance of these findings is discussed.     

INDUCTION OF APOGAMY IN MEGAGAMETOPHYTES OF ZAMIA INTEGRIFOLIA

Ovules of Zamia integrifolia Ait. were dissected and megagametophytes and embryos excised and cultured. Induction of apogamous, haploid roots and leaves by auxin and kinetin, in combination with glutamine, asparagine and alanine, was observed. The organs resembled their normal diploid counterparts. 2,4-D at 1 ppm was more effective than 1 ppm IAA, but less effective than 5 ppm IAA. The highest percentage of apogamy was 59% after 5 months of culture. In several cultures embryoids were formed both by diploid excised embryos and by haploid megagametophtyes.     

Regeneration of plantlets from in vitro raised leaf explants of Cleisostoma racimeferum Lindl

Protocorm like bodies (PLBs), callus and shoot buds developed in culture from in vitro raised foliar explants of Cleisostoma racimeferum. Among the different basal media, better result was obtained on MS medium containing sucrose (3%) and BA (2 microM) with approximately 80% frequency after 40 days of culture. Young leaves (15 week old) produced better PLBs. Whole leaf placed vertically upside-up orientation can regenerate PLBs and shoot buds (80%). PLBs and shoot buds formed on entire surface of the leaves. Cultures on BA and NAA (2 and 2 microM respectively in combination) stimulated callus mediated regeneration (68%). The rooted plantlets regenerated within 8-10 week from PLBs and shoot buds on MS medium containing IAA and kinetin (2 microM each in combination). BA containing medium triggered multiple shoot bud formation, while NAA alone or in combination with other growth regulators was inhibitory. Incorporation of activated charcoal (0.01%) in the medium stimulated formation of repetitive PLBs and multiple shoot buds. Rooted plants were ready for harvest after 20-22 week of initiation of culture. About 65% of the potted plants survived after 3 months in the poly house.     

GROWTH REGULATOR EFFECTS ON BUD INITIATION IN CALLUS CULTURES OF MEDICAGO SATIVA

Influence of growth regulators on bud initiation in callus of alfalfa (Medicago sativa L.) was studied by varying levels and combinations in the first medium of a two-medium sequence used to obtain whole plants. Callus of tetraploid clone S-4 (cv. ‘Saranac‘) was initiated from immature ovaries on a modified Blaydes' basal medium containing all combinations of six concentrations (0–36 μM) of kinetin (K), six concentrations (0–44 μM) of naphthaleneacetic acid (NAA), and seven concentrations (0–36 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D). After 28 days the callus was challenged to form buds by transfer to the modified Blaydes' medium containing 2.0 g/liter yeast extract and 0.57 mM inositol. No buds were produced in the absence of 2,4-D in the first medium, and the frequency of bud formation on the second medium was directly proportional to the 2,4-D concentration in the range 2.3–54 μM in the preceding medium. Buds were produced in the absence of kinetin in the first medium, but its presence in the range 2.3–36 μM markedly increased bud formation. NAA was not required for bud formation, and the budding frequency increased only slightly with increasing NAA concentration in the first medium. Budding of callus of two other alfalfa clones was also influenced by the 2,4-D concentration in the initial medium. There were several indications that many of the buds were initiated on the first medium and completed development on the second medium. These included the differential effect on budding of combinations of 2,4-D, NAA and kinetin in the callus initiation medium, the specific media sequence required, and the presence of embryoids on the callus which after transfer to the yeast extract-inositol medium produced buds.     

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.     

Tissue culture inHaworthia

Effects of three different auxins and kinetin in various combinations on greening and redifferentiation of the callus ofHaworthia setata were investigated. All auxins at the concentration of 50 mg/l inhibited callus greening. NAA in combination with kinetin promoted both callus greening and production of redifferentiated shoots. Low concentrations of IAA without kinetin promoted redifferentiation of shoots, but not callus greening. Addition of 2,4-D completely inhibited both greening and redifferentiation regardless of the level of kinetin except for the effects on shoot formation in the medium with 0.1 mg/l 2,4-D added. The calluses with the highest chlorophyll content were observed in the medium containing 2.0 mg/l kinetin without any auxins or with 0.1 mg/l NAA added. Most frequent shoot redifferentiation was observed in the medium containing 0.1 mg/l IAA without kinetin (redifferentiation rate; 67%), followed by the medium containing 10 mg/l NAA with 2.0 mg/l kinetin (44%), and 0.1 mg/l 2,4-D with 0.2 mg/l kinetin (33%). Generally, higher degrees of greening were associated with better growth. However, the auxins (IAA, NAA and 2,4-D) given at concentrations optimal for growth did not exhibit the highest degree of callus greening. Differences of the three auxins in their actions and interaction with kinetin were disclosed. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 423     

THE INITIATION OF SPOROPHYTES BY OBLIGATE APOGAMY IN CHEILANTHES CASTANEA

The initiation of apogamous sporophytes in Cheilanthes castanea was recorded by daily photography of individual gametophytes. Whereas an ordinary embryo arises from a zygote, apogamous embryos of C. castanea originate from one to three initial cells which occur just behind the apical region of the prothallus. The initial (or initials) produce cells with small chloroplasts behind the sinus of the gametophyte. The appearance of cells with smaller chloroplasts than those normally found in gametophytes is the first indication that apogamy is occurring. The cells with small plastids produce a group of densely-cytoplasmic meristematic cells. The size of the meristematic mass increases until shoot and root apices of the apogamous embryo are organized.     

Kinetin and naphtaleneacetic acid controlled starch formation in isolated roots ofPisunt sativum

The formation of starch in excised 3-days-old pea roots was enhanced up to three fold after cultivation for five days in liquid media containing 3% glucose, microelements, and 10^-6 M kinetin or 10^-7 M naphtaleneacetic acid (NAA). A synergistic effect of kinetin and NAA on starch formation was observed when both hormones were applied simultaneously over a wide range of concentrations.     

Influence of metabolic stress on the inheritance of cell determination in the moss, Pottia intermedia

Epigenetically-determined apogamy in aposporous regenerants of the moss Pottia intermedia persists during vegetative propagation, the capacity of apogamy being inherited by individual aposporous protonemal cells. To test Bauer-Lazarenko's proposal that stable apogamy in mosses may be due to some self-replicating cytoplasmic factor, the effects of different metabolic stress treatments on the expression of apogamy have been tested. Chronic metabolic stress caused by long-term growth of autotrophic aposporous protonema on mineral medium with 0.25% of casamino acids and on Murashiga-Skoog (MS) medium with sucrose and phytohormones, as well as by transitory action of high kinetin concentration, have a much stronger influence on the expression of apogamy, than short-term stress treatments with RNase and Pb(2+). Apogamy has been found to be lost stably, after prolonged growth on MS medium containing kinetin and ABA. The proposal that the capacity for apogamy is related to the release of aposporous protonemal cells from a putative factor for apogamy is discussed.     

Regulation of Organ Formation by Cytokinin and Auxin in Lilium Bulbscales Grown In Vitro

Regulation of organ formation by cytokinin and auxin was investigatedin vitro using Lilium auratum Lindl. (wild species habituatedin Japan) and Lilium speciosum Thunb. cv. "Uchida". The interactionof -naphthylacetic acid (NAA) and kinetin on bulbscale or rootdifferentiation was examined. NAA and kinetin showed mainlyindividual but also some synergistic effects. The effects ofbenzyladenine (BA) and kinetin were compared and the resultindicated that BA has a stronger physiological effect on organformation than kinetin and that their effects on Lilium auratumand Lilium speciosum were BA or kinetin-specific. The actionof kinetin on Lilium was affected by sucrose concentration andthe strength of the Murashige and Skoog medium (MS medium),and thus their high concentrations inhibited the kinetin-inducedbulbscale differentiation. Furthermore, a high sucrose levelnegated the kinetin inhibition of root formation, while highMS medium strength in itself inhibited root formation. Morphologicalobservation of bulbscale differentiation induced under a highkinetin level revealed that the new-formed structures are homologousto normally grown bulbs in soil in spite of their particularfeatures. (Received August 3, 1981; Accepted November 2, 1981)     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

影响籼稻体细胞胚胎发生几个因素的研究

以 IR36、IR50、IR52及 IR54等品种的幼穗及成熟种子为材料,研究了蔗糖浓度、2,4-D、NAA、激动素及脱落酸对体细胞胚胎发生、结构的保持及植株分化的影响。6%蔗糖有利于胚性愈伤组织的诱导;3%的有利于胚性结构的保持及植株分化。当培养基中不含2,4-D,而含激动素与 NAA 时,幼穗直接出芽;当不含激动素而含2,4-D与 NAA 时,外植体产生非胚性愈伤组织;当不含 NAA 而含2,4-D 与激动素时,外植体产生胚性愈伤组织。认为,2,4-D与激动素是籼稻体细胞胚胎发生的基本因素,而 NAA 的作用是不明显的。不同外植体(幼穗与成熟种子)的体细胞胚胎发生,对2,4-D 与激动素的反应略有不同,幼穗更为敏感。在继代培养基中,加入低浓度的脱落酸有利于胚性结构的保持。随着继代世代的延续,分化培养中愈伤组织所表现出的绿色生长点状物不能发育成完整植株。     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

硝酸镧对霍霍巴多芽苗生长的促进作用及植株再生

在增殖培养基(改良MS 2mg／L6-BA 0．5mg／LG3)中添加1～3．5mg／L硝酸镧,对霍霍巴试管苗生长有显著促进作用,2．5mg／L硝酸镧对多芽分化有极显著的促进;在生根培养基(改良1/2MS 3mg／LIBA 1．5mg／LNAA)中,1～2mg／L的La(NO3)3对根的分化有显著的促进作用,生根率提高,最佳浓度为2．0mg／L。过高的浓度对试管苗生长有一定抑制。试验表明,不同器官对硝酸镧的敏感程度不同。取2cm高以上的霍霍巴试管苗,用25mg／L IBA或NAA处理30min,扦插于沙基质中。保持室温20～30℃。50d后揭去覆膜,保持光强3000 lx,相对湿度85％以上。正常管理条件下成活率达75％。     

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. (c) 1993 John Wiley & Sons, Inc.     

Coumarin effects on Glycine max hypocotyl explants

Coumarin induced root formation and stimulated fresh weight production in hypocotyl explants of Glycine max L. cultured in vitro. All stimulatory effects caused by coumarin were induced within a relatively narrow range of concentrations between 1–500 μM, yielding optimum dose response curves. When coumarin was combined with kinetin fresh weight increased considerably, at optimum concentrations to a level almost as high as that obtained with NAA (10 μM) and kinetin (10 μM). Root formation was almost completely inhibited when kinetin was added in combination with coumarin. NAA + coumarin had small stimulatory effects on fresh weight. but were inhibitory in root formation. The frequency of rooting per explant, texture and pigmentation were also affected by different treatments.