Somatic embryogenesis and plant regeneration in Sorghum bicolor (L.) Moench

Summary Callus induction, somatic embryogenesis and plant regeneration were obtained in two cultivars of Sorghum bicolor (L.) Moench. Transverse thin cell layers from roots/epicotyls of 15-day-old seedlings or of regenerated plantlets were used. Callus response depended on the genotype, the size of transverse thin cell layers, the level at which transverse thin cell layers were excised on the epicotyl, the composition of growth substances and the number of in vitro regeneration cycles undergone by the donor plant. Somatic embryos were differentiated under a defined dark/light sequence, from epidermised compact calluses (i.e having already differentiated an epidermis), obtained directly with dicamba or from friable callus initiated with kinetin and 2,4 dichlorophenoxyacetic acid. The importance of kinetin and dicamba on the induction of embryogenic potential is reported.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - 2iP N6-(2-isopentyl)adenine - BAP 6-benzylaminopurine - CaMV cauliflower mosaïc virus - CPPU N-(2-chloro 4-pyridyl)-N-phenylurea - dicamba 3,6-dichloro-o-anisic acid - IAA indole-3-acetic acid - K kinetin - MS Murashige and Skoog - NAA -naphthaleneacetic acid - PEPC phosphoenolpyruvate carboxylase - SD standard deviation - tTCL transverse thin cell layer     

Genotypic and media effects on plant regeneration from cotyledon explant cultures of someBrassica species

Conditions were established for efficient plant regeneration from cotyledon explant calli in different cultivars ofBrassica juncea, B. campestris andB. carinata on Murashige & Skoog's (MS) medium supplemented with various combinations of cytokinins and auxins. Regeneration frequency, however, varied with genotype and the different growth hormone combinations in media. Almost in all species, MS medium with zeatin (1.0 mg 1^-1) and IAA (0.1 mg l^-1) was found to be best for shoot organogenesis followed by the ones containing high kinetin (2.0 mg l^-1) and low IAA (0.02 or 0.2 mg l^-1) concentrations. On these media, the cotyledonary explants invariably underwent callusing followed by multiple shoot formation, which could be separated and subcultured for further propagation. Number of shoots per cotyledon explant cultured varied from 0 to as many as 50. InB. juncea andB. campestris, the regeneration frequency declined sharply in the absence of auxin in medium. BAP in combination with NAA yielded no or a reduced number of shoots. Shoot organogenesis also declined with the reduction in photoperiod from continuous light to 16 hours. Shoots were easily rooted during prolonged incubation on the same medium and whole plants were transferred to pots in the greenhouse and grown to maturity.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - MS medium after Murashige & Skoog [8] - NAA -napthaleneacetic acid - ZEA Zeatin     

Improved plant regeneration in callus cultures of Sorghum bicolor (L.) Moench

Sorghum bicolor is a recalcitrant species for tissue culture regeneration and genetic transformation. Browning of explants is one of the factors limiting organ and tissue cultures. To overcome this, callus tissue was initiated from the shoot tips of in vitro germinating seeds (S. bicolor cv. Róna 1), and then cultured on modified MS media (Murashige and Skoog in Physiol Plant 15:473–497, 1962). In the first experiment, we tested callus induction on several media supplemented with casein hydrolysate, polyvinylpyrrolidone, honey, and sucrose. The best callus induction was recorded for the medium with honey and sucrose (80.0%) and for control medium (79.8%). Shoot regeneration was tested on the MS medium with 6-benzylaminopurine (BAP) supplemented with honey and sucrose at a 1:1 ratio (by weight) or with sucrose only. The highest percentage of calluses regenerating shoots was noted for those induced on the medium with sucrose and honey—approx. four times higher when compared to the control. Rooted plantlets were acclimatized with a 92% survival rate. In the second experiment, we analyzed culture responses to various ways of honey application to the induction media: honey (autoclaved or filtered) in presence or absence of sucrose. Supplementation of the medium with fructose, glucose, and maltose at a proportion typical for honey was also investigated. The explant and callus survival rates were similar to those of the honey–sucrose combination in the first experiment. Only presence of both sucrose and honey in the induction medium improved the total regeneration rate to 37.9% over the control (18.8%). Sucrose and honey appear to act synergistically for shoot regeneration in callus cultures of sorghum.

     

Somatic embryogenesis and plant regeneration of pepper in liquid media

A protocol was developed for regeneration of pepper (Capsicum annuum var. Ace) through somatic embryogenesis in liquid media. For embryogenic callus formation, mature zygotic embryo explants were used on basal Murashige and Skoog medium with 9.05 M 2,4-dichlorophenoxyacetic acid and 3% sucrose. Embryogenic callus was transferred to liquid basal Murashige and Skoog medium with 4.52 M 2,4-dichlorophenoxyacetic acid and 3% sucrose in order to increase the mass of the embryogenic culture. After pretreatment with potassium citrate, cells were placed into embryo initiation medium with 6 g l^-1 l-proline and a decreased (10 mM) ammonium concentration. Embryos were matured in 1.89 M abscisic acid containing half-strength Murashige and Skoog medium and converted into plants bothin vivo andin vitro at up to a 97% efficiency.     

连钱草组培快繁技术研究

用连钱草无菌茎尖为外植体进行快速繁殖,分别诱导、分化、生根形成再生植株进行快速繁殖,并移栽成活。结果表明在MS+6-BA1.5mg/L+NAA0.1mg/L培养基上诱导丛生芽效果最佳。在MS+IBA1.0mg/l+KT1.0mg/L培养基中根的诱导率为100%。     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Climate change and plant regeneration from seed

At the core of plant regeneration, temperature and water supply are critical drivers for seed dormancy (initiation, break) and germination. Hence, global climate change is altering these environmental cues and will preclude, delay, or enhance regeneration from seeds, as already documented in some cases. Along with compromised seedling emergence and vigour, shifts in germination phenology will influence population dynamics, and thus, species composition and diversity of communities. Altered seed maturation (including consequences for dispersal) and seed mass will have ramifications on life history traits of plants. Predicted changes in temperature and precipitation, and thus in soil moisture, will affect many components of seed persistence in soil, e.g. seed longevity, dormancy release and germination, and soil pathogen activity. More/less equitable climate will alter geographic distribution for species, but restricted migratory capacity in some will greatly limit their response. Seed traits for weedy species could evolve relatively quickly to keep pace with climate change enhancing their negative environmental and economic impact. Thus, increased research in understudied ecosystems, on key issues related to seed ecology, and on evolution of seed traits in nonweedy species is needed to more fully comprehend and plan for plant responses to global warming.     

Callus induction and plant regeneration from gladiolus

A method for the initiation of callus capable of plant regeneration from in vivo grown cormels of gladiolus (Gladiolus x grandiflorus Hort.) is described. Sliced cormels of the large-flowering hybrid, Peter Pears were cultured in vitro on a modified Murashige and Skoog medium, supplemented with various auxins. Yellow callus, which was either friable or compact, could be induced on all media tested. Callus induced on media with naphthaleneacetic acid failed to proliferate. Callus induced on media with 9 mM 2,4-dichlorophenoxyacetic acid showed the best growth. Addition of micro-elements and vitamins increased the induction and growth of callus capable of plant regeneration. Explants taken from the middle part of the cormels had the highest competence for callus initiation. Callus was induced on several gladiolus hybrids and the South African species G. garnierii Klatt. Callus induction was genotype dependent and among the cultivars tested, Peter Pears and White Prosperity were superior with respect to callus production on the media with either 2,4-dichlorophenoxyacetic acid or picloram. Plants were regenerated from yellow compact callus of all genotypes on media containing zeatin and benzyladenine in various concentrations.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog basal salt and vitamins (1962) - CI callus induction medium - NAA -naphthaleneacetic acid - BA 6-benzyladenine - picloram 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid - zeatin 6-[4-hydroxy-3-methylbut-2-enylamino]purine     

Direct plant regeneration from cucumber embryonal axis

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 μM BA and 1.59 μM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 μM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 μM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.     

Rapid plant regeneration from Nicotiana mesophyll protoplasts

A procedure is described for rapid plant regeneration from tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Six to seven days after protoplast isolation, colonies are placed on double filter feeder plates that consist of a strong regeneration medium containing 7.5 mg/l 6-(γ,γ-dimethylallylamino)-purine (2iP) and 0.1 mg/l p-chlorophenoxyacetic acid (pCPA). Complete plants are regenerated in about 5 weeks after transfer to a rooting medium (hormone-free Murashige and Skoog (MS) medium). However, upon remaining on shoot regeneration medium, 50–75 shoots are regenerated from single colonies derived from individual protoplasts. This procedure may reduce the amount of somaclonal variation (as measured by ploidy level) which is usually expressed in plants obtained by conventional regeneration techniques.     

Increased doubled haploid plant regeneration from rice (Oryza sativa L.) anthers cultured on colchicine-supplemented media

Plating rice anthers on a semisolid induction medium containing 250 or 500 mg/l colchicine for 24 or 48 h-incubations followed by transfer to colchicine-free medium and standard anther culture procedures resulted in overall 1.5- to 2.5- fold increases in doubled haploid green plant productions compared to control anther cultures. The addition of colchicine had no detrimental effects on the different anther culture efficiency parameters, but in some treatments led to significant enhancement of anther callusing frequency or callus green plant regenerating ability. The most efficient treatment raised doubled haploid plant recovery from 31% to 65.5%. These results suggest that post-plating colchicine treatment of anthers, since it was found to improve both anther culture efficiency and doubled haploid plant recovery frequency, could be integrated into rice doubled haploid plant production programmes.Abbreviations DH doubled haploid - NAA naphthalenacetic acid - PAS periodic acid Schiff     

Enhanced plant regeneration from microspore-derived embryos of Brassica napus by chilling,partial desiccation and age selection

Germination was readily induced in recalcitrant microspore-derived embryos of Brassica napus Topas when they were exposed to a period of chilling (9–12 days at 4°C) or partial desiccation (rapid or slow air drying) prior to germination. In general, embryos thirty-five days old had the highest germination rates as compared to younger or older ones. Populations of embryos were induced to germinate at a rate of over 90% under specific temperature, desiccation and age conditions. Comparisons to an embryogenic B. napus winter line, F346, are made.     

Ethylene influences green plant regeneration from barley callus

The plant hormone ethylene is involved in numerous plant processes including in vitro growth and regeneration. Manipulating ethylene in vitro may be useful for increasing plant regeneration from cultured cells. As part of ongoing efforts to improve plant regeneration from barley (Hordeum vulgare L.), we investigated ethylene emanation using our improved system and investigated methods of manipulating ethylene to increase regeneration. In vitro assays of regeneration from six cultivars, involving 10 weeks of callus initiation and proliferation followed by 8 weeks of plant regeneration, showed a correlation between regeneration and ethylene production: ethylene production was highest from ‘Golden Promise’, the best regenerator, and lowest from ‘Morex’ and ‘DH-20’, the poorest regenerators. Increasing ethylene production by addition of 1-aminocyclopropane 1-carboxylic acid (ACC) during weeks 8–10 increased regeneration from Morex. In contrast, adding ACC to Golden Promise cultures during any of the tissue culture steps reduced regeneration, suggesting that Golden Promise may produce more ethylene than needed for maximum regeneration rates. Blocking ethylene action with silver nitrate during weeks 5–10 almost doubled the regeneration from Morex and increased the Golden Promise regeneration 1.5-fold. Silver nitrate treatment of Golden Promise cultures during weeks 8–14 more than doubled the green plant regeneration. These results indicate that differential ethylene production is related to regeneration in the improved barley tissue culture system. Specific manipulations of ethylene were identified that can be used to increase the green plant regeneration from barley cultivars. The timing of ethylene action appears to be critical for maximum regeneration.     

Efficient plant regeneration from shoot apices of sorghum

An efficient and rapid regeneration protocol was developed using shoot apices from germinating seedlings of two cultivars of sorghum, SPV-462 and M35-1, as explants. A vertical slit given from the base of each dissected apex enhanced the efficiency of callusing response by two fold. MS medium containing 0.5 mg dm⁻³ each of 2,4-D and kinetin was most effective in producing friable and embryogenic calli. Scanning electron microscopy of these calli detected somatic embryogenesis. Calli thus induced gave rise to approximately 42 green shoots per callus in both the genotypes when transferred to regeneration medium containing 1.5 mg dm⁻³ kinetin.     

Plant regeneration from cultured immature embryos of Sorghum bicolor (L.) Moench

Summary Immature embryos of 20 sorghum genotypes were cultured on MS 5 medium containing MS mineral salts supplemented with 2,4-D, zeatin, glycine, niacinamide, Ca-pantothenate, L-asparagine, and vitamins. For regeneration, calli were transferred onto the same medium with the exception that IAA was substituted for 2,4-D. In general, immature embryos obtained 9–12 days after pollination resulted in the best redifferentiation. Ability of calli to regenerate varied among genotypes; cultivars C401-1 and C625 had the highest redifferentiation frequencies. Ability to redifferentiate was heritable and acted as a dominant trait. At least two gene pairs were involved. Regenerated R₀ plants were planted in a greenhouse and their selfed (R₁ and R₂) progenies were planted in the field and examined for morphological and cytological variations. The majority of the phenotypic variations noted in R₀ were not transmitted to later generations. However, variants for plant height, degree of fertility, and midrib color persisted in R₁ and R₂ generations. A variation in tallness was attributable to one dominant mutant gene. Short stature and male sterility variants appeared to be consequences of recessive mutant genes controlling those traits. Minor variations in peroxidase banding patterns were found among R₀ plants.This study was supported by a research grant from Kansas Sorghum Commission and by a Research Fellowship to the senior author from the Ministry of Agriculture, Animal Husbandry, and Fisheries, China. Contribution 86-456-J from the Kansas Agricultural Experiment Station     

Rapid plant regeneration from various explants of Jatropha integerrima

A simple, rapid and reproducible protocol for direct shoot regeneration from different explants of Jatropha integerrima was developed. Prolific adventitious shoot bud initiation was obtained using a combination of 2.2 or 4.4 M benzyladenine and 4.9 M indole-3-butyric acid (IBA). Reduction of IBA concentration (2.5 M) promoted further development of shoots. Regenerated shoots rooted readily on Murashige and Skoog (MS) medium lacking growth regulators. Plantlets were acclimatized and successfully transferred to pots.     

Prolific direct plant regeneration from cotyledons of white clover

Summary A facile procedure has been developed to regenerate white clover (Trifolium repens L.) plants, rapidly and directly from cotyledon explants of 3 day old seedlings. Scanning electron microscopy and histological sectioning demonstrated that shoot meristems developed from individual epidermal cells on the adaxial surface of the cotyledonary stalk, proximal to the site of excision. Initial cell divisions occurred after 2 days of culture and regenerated plants were transferred to soil within 6–8 weeks. Regenerated plants were normal, flowered and set seed. The highest shoot regeneration frequency (an average of 20 shoots per cotyledon) was obtained using an MS based medium containing 1.0 mg 1^-1 6-benzylaminopurine and 0.05 mg 1^-1 -napthaleneacetic acid. A similar regeneration frequency was obtained from cotyledon explants taken from eight different white clover cultivars.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - MS Murashige and Skoog medium     

Rapid plant regeneration from callus cultures of Plumbago zeylanica

Rapid plant regeneration was achieved in callus cultures derived from leaf and stem explants of Plumbago zeylanica Linn. on MS basal medium supplemented with 4.44 M 6-BA, 1.42 M IAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of growth regulators in the nutrient media. The leaf explants were more responsive (82.3%) than the stem explants on medium containing 1.42M IAA in combination with 4.44 M BA. The rate of regeneration was found to maintain the same level for 12 months without loss of vigour. Rooting of the differentiated shoots was achieved in media having 0.57 M IAA with 2% (w/v) sucrose within 10 days of culture. Regenerated plantlets were transferred to soil which grew normally with a survival rate of 90%. This protocol may help in the conservation of the species and selection of variants that may be induced to widen the genetic base of the genus.     

Callus induction and plant regeneration from maize mature embryos

Calli were induced from mature embryos of maize (Zea mays L.) inbred lines A632, B73 and Mol7 on MS medium supplemented with 1–2 mg/1 2,4-dichlorophenoxyacetic acid. Callus induction frequency ranged from 23–100%, with Mol7 having the highest frequency. Plants were regenerated from 4–5% of the B73 and Mol7 explants. Embryogenic and organogenic calli of B73 were maintained for more than two and one half years without losing regenerability. Of 95 regenerated plants, only one R₀ plant with abnormal pollen was detected, and no morphological variants were observed in the R₁ progeny.Abbreviations Dicamba 3,6-Dichloro-o-anisic acid - IAA 3-indoleacetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Ze zeatin     

Long-duration,high-frequency plant regeneration from cereal tissue cultures

By visual examination of calli derived from germinating seeds of wheat, oats, rice, proso millet, and pearl millet it has been possible to visually select embryogenic (E) callus which, on transfer to a regeneration medium, forms plants an average of 33 times more frequently than non-embryogenic (NE) callus of equal mass. Embryogenic callus consists of small isodiametric cells averaging 31 m in diameter; NE callus consists of long tubular cells averaging 52 m in width and 355 m in length. Production of E callus is in many cases promoted by media containing 2,4-di- or 2,4,5-trichlorophenoxyacetic acid (2,4-D or 2,4,5-T) plus indole-3-acetic acid or tryptophan+kinetin. Production on NE callus is promoted by media containing 2,4-D or 2,4,5-T alone. As a result of initial experiments to optimize both media for E callus production and media for plant regeneration, callus derived in six passages from an average of 26 seeds could produce about 1,000 regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - Trp L-tryptophan - E embryogenic - NE non-embryogenic