Plant regeneration fromBrassicanigra (L.) Koch stem protoplasts

Summary Protoplasts ofBrassica nigra (L.) Koch were isolated from stem peels of bolting racemes and cultured in 1.5 ml of VN1 liquid medium. The protoplasts in the liquid medium were plated on top of half strength MS medium supplemented with 400 mg/liter glutamine, 15 mg/liter glutathione, 50 mg/literl-serine, 0.25 mg/liter 6-benzylaminopurine, 0.5 mg/liter 2,4-dichlorophenoxyacetic acid, 1.5% sucrose, and 5% mannitol, pH, 5.7, solidified with 0.3% agarose. Ten percent of calli obtained from the protoplasts developed into plantlets within 4 wk after transfer onto 2N regeneration medium which contains MS salts plus 200 mg/liter casein hydrolysate, 0.625 mg/liter 6-benzylaminopurine, 0.625 mg/liter kinetin, 0.625 mg/liter 6-(γ,γ-dimethylallylamino)-purine, 0.625 mg/liter zeatin, 0.5 mg/liter 1-naphthaleneacetic acid, 1.5% sucrose, and 0.4% agarose. THis is the first report of plant regeneration fromB. nigra protoplasts.     

THE UPTAKE OF GLUCOSE BY CHLAMYDOMONAS SP.12

The glucose uptake of a species of Chlamydomonas was studied at various concentrations of d -glucose plus glucose-1-¹⁴C (0.003–10.0 mg/liter) and at various light levels (0–220 ft-c). The alga grows at 4 C either in the light or in the dark with added glucose, cellobiose, maltose, or fructose. Uptake of glucose could be described by the Michaelis-Menten equation, and both the maximum velocity of uptake and the half-saturation constant increased when the cells were exposed to glucose in the dark. However, the high value of the half-saturation constant (5 mg glucose/liter) compared with the low levels of glucose in nature (5–10 μg/liter) makes it unlikely that a transport system is effective under natural conditions. Even if a total of 10.0 mg/liter of glucose plus other organic compounds were available as substrate, the rate of photosynthesis would still be more than 10 times higher (at 220 ft-c) than the rate of organic substrate uptake. Light had no effect on the total uptake of glucose but did reduce the percentage of ¹⁴CO₂ evolved from 61% of the total ¹⁴CO taken up in the dark to 0% at 220 ft-c. This decrease could be due to either preferential use of the ¹⁴CO₂ in photosynthesis or of the photosynthate in respiration.     

加快棕种子萌发和幼苗生长的研究

棕种子萌发和幼苗生长十分缓慢 ,为了探索加快种子萌发和幼苗生长的方法 ,对棕种子进行不同处理、不同时间催芽和催芽后不同处理等试验 ,结果表明 :(1)生长调节剂 3号 ABT生根粉、吲哚丁酸和 2 ,4 - D可提高种子萌发率 ,促进幼苗生长。 (2 )播种前催芽可促使种子提前萌发 ,延长幼苗生长期 ,使当年生苗更粗壮 ;贮藏 3个月后催芽可提高 10 %～ 15%的发芽率。 (3)催芽后用 50 mg/ L和 10 0 mg/ L的 3号 ABT生根粉、或者 10 0 mg/ L的萘乙酸浸泡都可以提高幼苗出土率 ,但对幼苗生长没有明显影响。     

Establishment of suspension cultures from seeds of plains bluestem [Bothriochloa ischaemum (L.) Keng.] and regeneration of plants via somatic embryogenesis

Summary Mature seeds of plains Old World bluestem [Bothriochloa ischaemum (L.) Keng.] were used to initiate suspension cultures. The medium contained the major and minor minerals of Murashige and Skoog, Gamborg's B-5 vitamins, 30 g/liter sucrose, and 3 mg/liter 2,4-dichlorophenoxyacetic acid with or without 12 mM proline at pH 5.5. Cultures contained both embryogenic and nonembryogenic (NE) cells. Suspensions that had been filtered through a 40-mesh sieve were plated out on medium with 6 g/liter agar. Two-to-three weeks later, clumps that formed in suspension cultures that had been filtered previously were removed by filtration through a 40-mesh sieve and plated out on agar medium. Colonies were rated on the basis of surface area. of the total area of colonies formed from plated suspensions 70.9% were embryogenic, 19.8% were NE, and 9.3% were mixed colonies. Of the total area from plated clumps, 57.1% were E, 12.9% were NE, and 30% were mixed colonies. Embryoid maturation and germination was accomplished by transferring E or mixed colonies to MS medium with 1 mg/liter zeatin (mixted isomers). Rooting was completed on half-strength basal MS medium. Over 90% of plantlets survived transfer to the greenhouse and 95% of them survived transfer to the field. Seeds were provided by Dr. Charles Taliaferro, Agronomy Department, Oklahoma State University.     

AKINETE GERMINATION IN ANABAENA CIRCINALIS (CYANOPHTA)

Akinetes of a clonal culture of Anabaena circinalis Rabenhorst from Mt. Bold reservoir (eutrophic), South Australia, were isolated and the effects of light, phosphorus, and nitrogen availability on their germination were investigated. Light was required but there was no significant difference in percentage of germination after 72 h if akinetes were incubated in ASM-1 medium at irradiances of 15, 30, or 50 μmol.m^-2.s^-1. Maximum akinete germination occurred by 48 h. Nitrogen was not required, as 88% of akinetes germinated in the flasks without combined nitrogen added to the medium and without N₂ in the air. NH₄⁺-N at 28 mg N.L^-1 completely suppressed germination, whereas 28 mg NO₃ N.L^-1 had no effect relative to the controls without nitrogen. Phosphorus was required, and at 48 h percentage of germination in the flasks with 0.6 mg P.L^-1 added (78%) was significantly greater than in the flasks with 0.06 P.L^-1 (58%) and 0 mgP.L^-1 (24%) added. Germlings in the 0 mg P.L^-1 flasks were only 2–4 cells long and stunted in appearance, whereas germlings at all other P concentrations were 8–16 cells long. It is likely that the isolation process exposed some akinetes to intracellular phosphorus released from lysing vegetative cells, but this was insufficient to allow normal development in the 0 mg P.L^-1 flasks. The plot of percentage of germination vs. initial phosphorus concentration, in the medium showed a relationship analogous to Michaelis-Menten nutrient uptake kinetics, suggesting that a specific membrane-bound enzyme system(s) is involved, with phosphorus as the substrate. The half saturation value (K_S) for germination was 50 μg P.L^-1.     

Post-harvest age-induced seed dormancy of Acer ginnala and its alleviation by growth regulator and low temperature treatments

One-month-old fruits of Acer ginnala with winged pericarp attached gave 44% germination and this was not increased by cold treatment at 4°C for 0, 10, 20, or 30 days, gibberellic acid treatment at 0, 1, 10, 100 or 1000 mg litre^-1, or ethephon treatment at 0, 2, 20, 200 or 2000 mg litre^-1. After 6 months of storage at 20–25 °C, germination of untreated fruits fell to 5% but could be restored to that of 1-month-old fruits by incubation at 4 °C for 30 days. After 9 months storage, no germination occurred in untreated fruits. Cold treatment (30 days at 4 °C partially restored germination (26%). Treatment with either gibberellic acid (1000 mg litre^-1) and 30 days at 4 °C (40%) or ethephon (100 mg litre^-] and 30 days at 4 °C improved germination (69%). The combination of all three treatments, i.e. 100 mg litre^-1 gibberellic acid, 100 mg litre^-1 ethephon and 30 days at 4 °C, optimised germination (86%). Thus, dormancy of A. ginnala developed during storage but could be reversed by a combination of treatment with low temperature and growth regulators. The highest germination (86%) was achieved after low temperature and growth regulator treatment of stored fruit.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

Sensitivity of thermophilic methanogenic bacteria to heavy metals

The effects of Cd, Cu, and Ni on pure cultures of thermophilic methanogenic bacteria were studied. The bacteria used wereMethanobacterium thermoautotrophicum and TAM, a thermophilic, acetate-decarboxylating, methanogenic bacterium. Much lower concentrations of heavy metals were needed to cause initial inhibition of TAM (1 mg/liter Cu and Cd; 5 mg/liter Ni) compared withM. thermoautotrophicum (10 mg/liter Cu and Cd; and 100 mg/liter Ni). No growth of TAM occurred at 5 mg/liter Cu and 25 mg/liter Ni, while the corresponding values forM. thermoautotrophicum were 50 mg/liter Cu and 200 mg/liter Ni. Cd (50 mg/liter) was totally inhibitory toM. thermoautotrophicum but allowed minimal growth of TAM. Ni stimulated both organisms at an optimal concentration of 5 mg/liter forM. thermoautotrophicum and 1 mg/liter for TAM. The toxicity of Cd and Cu was found to depend upon the presence of Ni in the medium.     

Organogenesis in leafy spurge (Euphorbia esula L.)

Summary All parts of leafy spurge seedlings can be regenerated when isolated and placed onto B5 medium. One-centimeter isolated hypocotyl segments were tested successfully for their usefulness as a bioassay system by comparing the response of auxins, herbicides, and cytokinins. Indole-3-acetic acid (IAA) was the most effective auxin to stimulate root formation. IAA was effective whether the hypocotyl segments remained on the same medium up to 60 days, or the segments were transferred to basal media after 2 or 5 days (pulse treatment). Pulse treatments with the other auxins resulted in stimulation of root formation; continuous or 5-day pulses of higher concentrations of indole-3-butyric acid,α-naphthaleneacetic acid and especially 2,4-dichlorophenoxyacetic acid and picloram formed excessive callus instead of roots. Picloram did not stimulate root formation, whether the treatment was continuous or pulse-treated. No roots formed with continuous picloram at 0.1 mg/liter or greater, but transfer to basal media did result in root and shoot formation at about 50% of the number formed on the controls. Lesser picloram concentrations had no effect. Shoots formed readily on untreated (control) segments, but continuous treatment with all three cytokinins, kinetin, zeatin, and zeatin riboside, increased the numbers of shoots about equally. Root formation was inhibited by the cytokinins at the higher concentrations (0.1 to 0.2 mg/liter). With the exception of a 5-day pulse of 0.04 mg/liter IAA, the auxins did not stimulate shoot formation, but generally inhibited shoot formation, even in pulse-treated cultures.     

Formation of higher alcohols and phenol by strains of zymomonas sp.

Strains of the bacteria Zymomonas sp. were studied for their ability to form higher alcohols. In a complex growth medium, six strains were shown to produce significant amounts of 1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, pentanols, secondary hexyl-alcohols, and trace amounts of n-hexanol. When resting cells of these organisms were placed into a fermentation medium containing glucose and Tris-buffer, Z. mobilis 8938 produced increased levels of 1-butanol, and secondary hexyl-alcohols at concentrations of 13.5 mg/liter and 5.8 mg/liter, respectively. Another strain, Z. mobilis subsp. mobilis B 806, stimulated the formation of 1-propanol and 1-butanol at concentrations of 14.9 mg/liter and 23.52 mg/liter, respectively. Amino acids or amino acid precursors were then added to the fermentation medium. The presence of threonine and α-ketobutyric acid stimulated Z. mobilis 8938 to produce 82.6 mg/liter secondary hexyl-alcohols and 8.0 mg/liter n-hexanol, respectively. Isoleucine and valine increased the production of 2-methyl-1-butanol (394.0 mg/liter) and 3-methyl-1-butanol (113.4 mg/liter), respectively, by Z. mobilis subsp. mobilis B 806. Glutamine enhanced the formation of 2-methyl-2-butanol production to concentrations 38.8 mg/liter in Zymomonas strain B 806. Additional experiments suggested that higher alcohol production could also be accomplished in the absence of glucose when cells were allowed to metabolize the precursors only. The effect of aromatic amino acids on phenol production was determined using resting cells of Zymomonas sp. The maximum yield of phenol (111.6 mg/liter) was found by Zymomonas strain 8938 in the presence of tyrosine. The addition of phenylalanine also stimulated this strain to form 71.4 mg/liter of phenol.     

In vitro shoot tip multiplication of cowpea Vigna unguiculata (L.) walp

Summary Shoot multiplication was induced in cowpea, cv. Georgia-21, from shoot tip explants. Shoot tips, 5 mm long, were isolated from in vitro-grown seedlings and cultured on MS medium containing N⁶-benzyladenine (BA) at 1, 2.5, or 5 mg/liter (4.4, 11.1, or 22.2 μM) or 6-furfurylaminopurine (kinetin) at 1, 2.5, or 5 mg/liter (4.6, 11.6, or 23.2 μM) combined with 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.3 μM) or naphthaleneacetic acid (NAA) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.7 μM). Cultures were maintained at a 12-h photoperiod (40 μmol·m⁻²·s⁻¹) and 23 ± 2° C. Treatments with BA induced greater shoot proliferation than those with kinetin. The highest number of shoots was produced on 5 mg (22.2 μM) BA per liter in combination with NAA or 2,4-D at 0.01 mg/liter (0.05 μM). Callus proliferated from the basal ends of shoot pieces in all treatments. The cultures also formed roots in the presence of kinetin, but not on BA-containing medium. To produce whole plants, the shoots were separated and rooted on 0.1 mg (0.5 μM) NAA per liter. Resulting plants grew normally under greenhouse conditions. Shoot tips provide an excellent explant source for cowpea micropropagation and can be used for callus induction.     

Effect of gibberellic acid- and water-based pre-soaking treatments under different temperatures and photoperiods on the seed germination of Allium stracheyi Baker: An endangered alpine species of Central Himalaya,India

Allium stracheyi Baker (Alliaceae, 2600–3000 m asl), an endangered species of Central Himalaya, India, has low seed germination in its natural habitat. This study is an attempt to improve seed germination by determining the seed viability with a low mean germination time (MGT) and germination index (GI) under optimum temperature, light, and pre-soaking treatments. The seeds were pre-soaked in hot water (80°C), cold water (10°C), and gibberellic acid (GA₃ at 50 and 100 mg/l) for 24 h and subjected to light (12 h light and 12 h dark) and continuous dark (24 h) conditions with different temperature regimes (10, 15, 20, 25, and 30°C). The viability varied between 66.0% and 69.67% and declined rapidly after 12 months of storage. Our studies suggest that the 100 mg/l GA₃ treatment was beneficial for seed germination and seedling growth. Pre-soaking in a 100 mg/l GA₃ solution and incubation at 20°C under light conditions enhanced the germination significantly (p < 0.05) and resulted in the highest (97.3%) germination with the lowest MGT = 5.7 days, with GI = 8.11. The recommendations of this study support the conservation of alpine A. stracheyi via simple and cost-effective techniques for optimal seed germination.     

Regeneration of garlic plants (allium sativum L., CV. “Chonan”) via cell culture in liquid medium

Summary Aiming at the genetic improvement of garlic cultivars, a cell suspension protocol was established which includes the induction of friable callus, establishment of cells in liquid medium, plating, regeneration, and bulb formation. Calluses of various textures from compact to friable and from green to yellowish were obtained by culturing explants excised from inner leaves of garlic bulbs on Marashig-Shoog (MS) medium with 2,4 dichlorophenoxy acetic acid (2,4-D), (1.1 mg/liter [5.0 μM]), picloram (1.2 mg/liter [5.0 μM]), and kinetin (2.1 mg/liter [10 μM]). Friable callus occurred on MS-A contained 2,4-D alone (1.0 mg/liter [4.52 μM]) and this callus was used to develop cell suspension cultures, which were maintained in liquid MS-B medium with a 2,4-D/benzyl adenine (BA) (0.5 mg/liter [2.25 μM]: 0.5 mg/liter [2.22 μM]) ratio. High plating efficiency was obtained on MS-C medium with different naphthalene acetic acid/BA combinations. Regeneration occurred after transfer of the caulogenic mass to MS-C medium containing 10 mg/liter (74.02 μM) and 20 mg/liter (148.04 μM) adenine for 60 days, followed by transfer to adenine-free medium. Plantlets transplanted to soil showed normal phenology. Shoots grown on modified MS medium supplemented with indolylbutryic acid (3.0 mg/liter [14.7 μM]) stimulated bulb formation by 30 days in culture.     

Toxicity of citric and succinic acids for the pycnidiospores ofBotryodiplodia theobromae

The toxic effect of citric and succinic acids on the germination of the pycnidiospores ofBotryodiplodia theobromae, mycelial growth and the killing rate of theB. theobromae spores was investigated. The percentage inhibition of germination of viable fungal spores by 0.01% succinic or citric acid ranged between 51.6 and 58.1%, respectively.B. theobromae was found to grow in 2% malt extract broth at 28°C at the rate of 0.13 CFU/h. Citric acid exhibited a higher killing rate of 0.26 CFU/h and was more effective against the germination of the fungal spores. At concentrations of 0.3% and above, citric acid could be used as pre- and post-infectional fungicide.     

Methyl Bromide Fumigation of Pratylenchus brachyurus in Peanut Shells

Five dosages of methyl bromide were used to fumigate peanut (Arachis hypogaea L.) shells and whole pods of peanuts in 1-liter flasks for 24 hr at 25 C. Methyl bromide dosages as low as 24.5 mg/liter killed all Pratylenchus brachyurus (Godfrey) Filip. &Sch. Stech. in peanut shells. Dosages of 44.6 and 50.9 mg/liter killed all but one or two nematodes in shells of whole pods. A 15% reduction in seed germination occurred at the 50.9-mg/liter dosage.     

Substrate,Hormone, Winnowing,and Stratification Influence the Seed Germination of <i>Ilex asprella</i> (Hook. et Arn.) Champ. ex Benth

Ilex asprella (Hook. et Arn.) Champ. ex Benth is one of the most important traditional Chinese medicines in southern China. The seeds of Ilex asprella usually have extremely low germination due to their dormancy characteristics, which severely impacts the efficiency of seedling raising and increases labor costs. In this study, to improve the seed germination of I. asprella, the effects of germination substrate, hormone, winnowing, and stratification treatments on the seed germination of I. asprella were investigated. The results of the germination matrix showed that the highest germination percentage of 45.2% was achieved under the 20°C/10°C day/night temperature and vermiculite germination medium conditions. The results of hormone treatments revealed that 100–400 mg/L of gibberellin (GA) and 50–100 mg/L of salicylic acid (SA) were found to be effective in releasing the dormancy of I. asprella seeds. Moreover, winnowing could effectively eliminate unsaturated seeds and impurities, thus improving the seed germination of I. asprella. Furthermore, warm temperature (15°C) stratification could expand the temperature range of I. asprella’s seed germination, which was beneficial for seed germination of I. asprella and for seed nursery at room temperature in production practice. The present study obtained a method to break dormancy and increase seed germination in I. asprella, thereby forming a groundwork for improving the efficiency of large-scale planting of I. asprella.     

Seed moisture content affects afterripening and smoke responsiveness in three sympatric Australian native species from fire‐prone environments

Germination of freshly collected seeds of three sympatric herbaceous species native to fire‐prone environments in south‐western Australia was significantly improved through the application of novel combinations of dry heat, gibberellic acid, smoke water and dry afterripening. For fresh seeds, combinations of dry heat, gibberellic acid and/or smoke water resulted in >80% germination in Austrostipa elegantissima (Poaceae) and Stylidium affine (Stylidaceae) seeds and >60% germination in Conostylis candicans (Haemodoraceae) seeds, compared with <10% germination of control seeds. For fresh seeds, two broad germination patterns were observed in response to smoke water: nil – low germination for both control and smoke water‐treated seeds (A. elegantissima and S. affine); and a significant smoke response (35%) compared with control seeds (1%) (C. candicans). During afterripening, high germination for A. elegantissima seeds was achieved following 3 months storage of seeds at equilibrium relative humidities of 23–75%, but seeds stored at 5–13% equilibrium relative humidities took 6–36 months to achieve similar levels of germination. Germination of C. candicans seeds also increased after 3 months storage, to >60% at each equilibrium relative humidity and further increases over time were slight. For S. affine seeds >60% germination was achieved only after 36 months storage at 50% equilibrium relative humidity. Seeds from all three species were smoke‐responsive at some point, but the interaction/effects of afterripening on the smoke response varied significantly between species. This study highlights an apparent effect of seed dormancy status on response to smoke and a surprisingly high level of ecological variation in pre‐germination requirements (cues) for these co‐occurring species that may relate to variation(s) in microsite selection forces operating on the soil seed bank of the different species.     

Plant regeneration from isolated cells ofLavandula latifolia medicus

Summary Single cells were obtained from hypocotyl-derived callus ofLavandula latifolia Medicus. Cells were plated in Murashige and Skoog medium supplemented with indoleacetic acid (IAA), benzyladenine (BA), and several IAA-BA combinations. Cell division required the simultaneous presence of IAA and BA in the culture medium, but callus formation was only achieved with 0.1 or 1 mg/liter IAA and 2 mg/liter BA. To induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on the composition of both the callus induction and the shoot regeneration media, best results being obtained when calli grown in 1 mg/liter IAA and 2 mg/liter BA were subcultured to media containing 2 mg/liter BA and 15% coconut milk. Under these conditions, up to 75% of calli formed shoots that subsequently were rooted and established in soil.     

Influence of an abscisic acid (ABA) seed coating on seed germination rate and timing of Bluebunch Wheatgrass

Semi‐arid rangeland degradation is a reoccurring issue throughout the world. In the Great Basin of North America, seeds sown in the fall to restore degraded sagebrush (Artemisia spp.) steppe plant communities may experience high mortality in winter due to exposure of seedlings to freezing temperatures and other stressors. Delaying germination until early spring when conditions are more suitable for growth may increase survival. We evaluated the use of BioNik? (Valent BioSciences LLC) abscisic acid (ABA) to delay germination of bluebunch wheatgrass (Pseudoroegneria spicata). Seed was either left untreated or coated at five separate rates of ABA ranging from 0.25 to 6.0 g 100 g^?1 of seed. Seeds were incubated at five separate constant temperatures from 5 to 25°C. From the resultant germination data, we developed quadratic thermal accumulation models for each treatment and applied them to 4 years of historic soil moisture and temperature data across six sagebrush steppe sites to predict germination timing. Total germination percentage remained similar across all temperatures except at 25°C, where high ABA rates had slightly lower values. All ABA doses delayed germination, with the greatest delays at 5–10°C. For example, the time required for 50% of the seeds to germinate at 5°C was increased by 16–46 d, depending on the amount of ABA applied. Seed germination models predicted that the majority of untreated seed would germinate 5–11 weeks after a 15 October simulated planting date. In contrast, seeds treated with ABA were predicted to delay germination to late winter or early spring. These results indicate that ABA coatings may delay germination of fall planted seed until conditions are more suitable for plant survival and growth.     

ATTEMPTS TO OBTAIN BACTERIA-FREE PLANTS OF PSYCHOTRIA PUNCTATA (RUBIACEAE): GROWTH AND ROOT FORMATION IN CALLUS CULTURES

Attempts were made to obtain bacteria-free plants of Psychotria punctata from tissue cultures. Stem explants and callus derived from them were induced to form roots but failed to form buds on Linsmaier and Skoog medium and 96 chemical modifications of it, including most of those known to induce bud formation in other species. Roots formed with ample IAA (2 mg/liter or more) and a low kinetin concentration (0.25 or 0.50 mg/liter). Adenine inhibited root formation in these media, but tyrosine did not. Tyrosine did lower the percentage of calluses commencing growth. When enzyme-hydrolyzed lactalbumin (1.3 g/liter), kinetin (0.5 mg/liter) and IAA (5 mg/liter) were added to Linsmaier and Skoog medium modified by decreasing inorganic nitrogen and increasing inorganic phosphate, callus grew at the fastest rate observed (increasing threefold in fresh weight in three weeks) and formed numerous roots. This was adopted as the stock callus medium. Casein hydrolysates also stimulated growth but less so than lactalbumin hydrolysate. When lactalbumin hydrolysate or a casein hydrolysate lacking tryptophan was supplied, growth occurred without added auxin if sufficient cytokinin was added. Cytokinin was required at unusually high concentration and was tolerated at still higher concentration. Formation, elongation, and branching of roots persisted on a saturated solution of BA which inhibited callus growth about 70 % and delayed callus senescence. Light caused earlier callus senescence after growth had ceased but did not affect callus growth or root formation. Light-induced senescence was prevented by a high cytokinin concentration.