An improvement of tomato protoplast culture for rapid plant regeneration

Tomato mesophyll protoplasts were cultured in TM2 medium containing 5.7 M -naphthaleneacetic acid and 2.4 M benzyladenine and were incubated either in stationary culture or on an orbital shaker at 25–30 strokes per min, in combination with interval addition of fresh medium. The effects of stationary and shaking conditions on the growth of the colonies and their subsequent shoot organogenesis were significantly different. The cultures maintained in stationary condition without adding fresh medium accumulated a thin membranous layer on the medium surface and whitish substance in the medium that seemed to precede cell browning and premature colony death. Mild shaking conditions along with the reduction of colony density by one half by dividing the contents of one dish into two dishes, after adding 2 ml of fresh medium on the 4th day and further addition of fresh medium (0.5 ml) on the 8th day of plating, provided optimal conditions for colony growth and suppressed thin layer and whitish substance accumulation. Ten-day-old colonies raised through this protocol regenerated shoots rapidly (within 19–20 days after initial plating) after transfer to regeneration medium (MS medium with 2.8 M zeatin riboside, 0.06–0.1 M gibberellic acid, 4% sucrose and 1% type VII agarose) directly bypassing the callus phase.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige & Skoog (1962) medium - NAA -naphthaleneacetic acid - SPM stroke per min - GLM General Linear Models - 2,4-D 2,4-dichlorophenoxyacetic acid     

Studies of protoplast culture and plant regeneration from commercial and rapid-cyclingBrassica species

Protoplasts were isolated from aseptic shoot cultures of commercial cultivars ofBrassica napus, B. oleracea andB. campestris, and from the six rapid-cycling brassica species. Of the rapid-cycling species, onlyB. napus responded well to the culture conditions used; 2% of protoplasts formed calli and up to 5% of calli regenerated shoots. Regeneration was also achieved from commercial cultivars ofB. napus andB. oleracea. For these two species the plating density, time of dilution with fresh medium and the composition of the shoot-inducing medium were all found to have an important influence on the efficiency of plant regeneration. Both responded better to maltose than to sucrose-based media. Under the optimum conditionsB. napus showed a plating efficiency of 7.8% and shooting efficiency of 17%; forB. oleracea the figures were 2% and 56%, respectively.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration and protoplast culture of Browollia speciosa

Explants from hypcotyls and cotyledons of Browalia speciosa were shown to regenerate plantlets.Protoplasts were isolated from etiolated cotyledon material, and, although callus was readily obtained, plantlet regeneration was not observed using numerous hormone regimes.Abbreviations M Mannitol - 2,4-D Dichlorophenoxyacetic acid - NAA Naphthalene-acetic acid - BAP Benzylaminopurine - MS medium Murashige and Skoog (1962) medium - UM medium Uchimiya and Murashige (1974) medium - COT cotyledon - SH shoot - R root     

Plastid number and plastid structural changes associated with tobacco mesophyll protoplast culture and plant regeneration

Mesophyll protoplasts were isolated from Nicotiana tabacum L. cv. Xanthi, and cell-colony formation induced in liquid culture. The plastid changes associated with the morphogenetic sequence from mesophyll protoplast to whole plant were examined. Minor ultrastructural changes in the plastids were evident after 1 d of culture, but by 8 d (four-to-eight-cell stage) the plastids were small, there was much less thylakoid membrane appression, and many prominent plastoglobuli were also present. Plastid-division figures were evident at this point of time and it was common to find plastids clustered around the nucleus. A typical proplastid was the dominant plastid type in the cultured cells from about 11 d until about five weeks when large amyloplasts and pregranal plastids were observed. Normally structured chloroplasts were present in the regenerated plant. There was no plastid division until the four-cell stage, with plastid numbers per cell approximately halving at each cell division, then stabilising around 12 per cell during cell-colony development, a number typical of meristematic cells. Though nucleoids were always present, their numbers in the plastids were reduced by the eight-cell stage.     

Plant regeneration from petal protoplast culture ofPetunia hybrida

Morphologically normal plants have been regenerated from petal protoplasts of petunia (Petunia hybrida) flower. Maximum protoplast yields from petal tissues were obtained within 2 days after anthesis. Protoplasts were cultured on modified Murashige and Skoog's medium in which NH₄NO₃ and Fe·EDTA concentrations were reduced to 1/3 (7mM) and 1/10 (10 M), respectively. After plating, protoplasts gradually reduced pigment density, and plastids developed near the nucleus. In premitotic petunia petal cells, the nucleus moved from the periphery to the central region of the cell. The first cell divisions were detected after 6–10 days of culture initiation, and the average division frequency was 15% in the best culture condition. The results indicated that the time of the first cell division and cell division frequency were closely related to flower age after anthesis. More than a hundred plants with morphologically normal shoots and roots have been obtained. Those plants grew vigorously in soil.Abbreviations BA benzylaminopurine - DAPI 4, 6-diamidino-2-phenylindole - 2, 4-d dichlorophenoxyacetic acid - Fe·EDTA Fe·ethylenediaminetetraacetate - IAA indole-3-acetic acid - MtSB microtubule stabilizing buffer - NAA -naphthaleneacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid     

植物原生质体培养研究进展

自从Ｃｏｃｋｉｎｇ(196 0 )用酶法首次分离出有活性的原生质体 ,1971年Ｔａｋｅｂｅ等首次从烟草叶片分离原生质体 ,经培养获得再生植株 ,原生质体的研究和应用进入了一个新阶段。1　原生质体培养影响因子的研究1 1　培养基种类及成分不同植物原生质体培养的基本培养基不尽相同。培养基种类影响到原生质体的分裂频率、植板率以及小愈伤组织的出现等[1,2 ] 。潘增光[3 ] 等比较了 4种培养基 (ＭＳ、ＭＴ、改良ＭＴ、ＢＨ3)对苹果叶肉原生质体培养的影响 ,结果表明 ,只有在改良ＭＴ培养基中能观察到多细胞团形成 ,而在ＭＳ、ＭＴ、ＢＨ3作为…     

Predictive correlates of shoot regeneration from potato protoplast culture

Summary Adaptation of protoplast regeneration systems for use on untested or recalcitrant potato genotypes can be a time-consuming exercise. Callus growth and xylogenesis were evaluated as early correlates of shooting potential to shorten this process. Callus growth was of limited value for predicting organogenesis but a linear relationship was observed between xylogenesis and shooting frequency. Increases in xylem content above a minimum threshold corresponded with increases in shooting frequency. The predictive value of the relationship was tested using a simple protocol modification (the culture of calli on a filter paper base). Calli on filter paper produced more xylem elements and shoots than those plated directly on medium. The potential of xylem content as a predictive test of shooting frequency is discussed.     

Shoot regeneration,re-flowering and post flowering survival in bamboo inflorescence culture

In vitro grown inflorescences of Bambusa edulis were used to investigate the process of vegetative shoot growth in detail. The findings revealed that auxins and ACC could be significant growth regulators in this process. Overall, auxins [NAA, indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)] induced inflorescences to grow vegetative shoots. However, the efficiency of shoot regeneration varied. A greater percentage (27.3–34.5) of inflorescences in the 5 mg l⁻¹ NAA, 10 mg l⁻¹ NAA, and 1 mg l⁻¹ 2,4-D treatments formed more vegetative shoots than those exposed to other treatments. IBA promoted shoot regeneration less effectively than NAA and 2,4-D. Fifty percent of regenerated vegetative shoots flowered after 2 months when the medium was supplemented with 5 mg l⁻¹ NAA. All shoots that received 1 mg l⁻¹ 1-amino-cyclopropane-1-carboxylic acid (ACC) flowered in 5 mg l⁻¹ NAA medium. Rooted plantlets were used to examine their survival following in vitro flowering. All plantlets with vegetative shoots, even those with inflorescences, survived and grew.     

Somatic embryogenesis and plant regeneration from immature inflorescence segments of Coix lacryma-jobi

Callus was obtained from segments of immature inflorescence of Coix lacryma-jobi cultured on N6 medium containing 1–2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 3–5% sucrose. Plantlets were regenerated when embryogenic calluses were transferred onto MS medium with 0.5 mg/l kinetin and 0.01 mg/l naphthaleneacetic acid (NAA). Regenerated plants had the diploid chromosome number (2n=20).     

亚麻原生质体培养及植株再生

Shoot protoplasts of four fiber flax (Linum usitatissimum) varieties (7309, 948, Belinka and Viking) were isolated and cultured. The optimal condition for higher protoplast yield 1.8 x 10(6)/gFW and activity 85.5% (c.v. 948) were from 10 day old seedings. Culture in V-KM Agroase-island medium led to first divisions after 3 days (c. v. 948), and after twenty days with an efficiency of 36% of divided cells and 5.2% in plating efficiency. Plant regeneration was obtained in 7309 and Belinka on agar media B5-2, MS3 containing 0.6 mg/L 6-BA and 0.1 mg/L NAA. Roots and leaves regeneration were observed in Viking and 948 respectively.     

Protoplast culture and plant regeneration ofPinellia ternata

A study was undertaken to develop a protoplast regeneration system for pinellia. A yield of 19 29 x 10⁵ protoplasts/g F. W. could be obtained from cell suspension cultures incubated in a digestion enzyme solution with 2% cellulase Onzuka R-10, 10% pectinase (Sigma), 0.01% pectolyase Y23. K8P and modified MS media were used to culture protoplasts in: a) liquid, b) liquid-solid double layer, or c) agarose embedded protoplast culture. The former two were conducive to colony formation from protoplast-derived cells. The frequency of cell division was about 8% after 3 days in culture. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Calli (1–2 mm in diameter) formed after 30–40 days in culture. The calli transferred onto medium supplemented with KT (0.5 mg 1^–1) and NAA (0.2 mg 1)^–1) could regenerate plants after 40–50 days. Of 47 plantlets transplanted into plots, 29 flowered and were fertile.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - KT kinetin - CH casein hydrolysate     

Studies of protoplast culture types and plant regeneration from callusderived protoplasts of Asparagus officinalis L. cv. Lucullus 234

Plating efficiency and colony formation of callus-derived protoplasts of Asparagus officinalis L. cv. Lucullus 234 differed significantly with different protoplast culture media and types of culture. Osmotic conditions and hormone concentrations of liquid media produced the greatest influence on plating efficiency and colony formation in bead culture. Protoplasts grew best in bead culture with a solid modified Kao & Michayluk protoplast culture medium (KM) supplemented with 0.5 mg l^–1 -naphthaleneacetic acid (NAA), 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg l^–1 kinetin, and 0.6% agarose (KM6) and a liquid modified KM medium differing from KM6 medium in sugar content, having 0.18 M sucrose and 0.18 M mannitol (A8). An average plating efficiency of 19.1% and colony formation of 15.5% was obtained one week after isolation in bead culture with the KM6 and A8 media. The highest average shoot regeneration of 92.3% was obtained with a Murashige & Skoog medium (MS) containing 0.125 mg l^–1 NAA, 0.125 mg l^–1 2,4-D, 0.25 mg l^–1 6-benzylaminopurine (6-BAP) and 3% sucrose. Plants have been regenerated and transferred to the greenhouse.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine     

Factors affecting plant regeneration from immature inflorescence of two winter wheat cultivars

Inflorescence explants of two winter wheat cultivars, Triticum durum cv. Kızıltan-91 and T. aestivum cv. Bezostaja-01, were used to evaluate the effects of vernalization period of donor plants, callus age and medium composition on regeneration capacity. Donor plants were grown for 7 d and they were exposed to 4 °C for 1, 2, 3, 4, and 5 weeks. The maximum inflorescence formation was observed as 79 % at 4 weeks and 73 % at 5 weeks of vernalization period for Kızıltan-91 and Bezostaja-01, respectively. Among 6 different callus induction and regeneration mediums, I₁-R₁ and I₃-R₃ have to be the best responding mediums for Kızıltan-91 and Bezostaja-01, respectively. In Kızıltan-91, calli induced from donor plants, vernalized for 3 weeks, showed a significantly lower regeneration capacity than counterparts vernalized for 4 and 5 weeks. The highest regeneration capacity of 69 % was obtained from 6-week-old calli produced from 4 weeks vernalized Kızıltan-91 donor plants. In contrast to Kızıltan-91 cultures, the effects of vernalization period and callus age on regeneration capacity were not significant in Bezostaja-01 cultures. The maximum numbers of tillers were obtained from 6-and 15-week-old calli for Bezostaja-01 and Kızıltan-91, respectively. In contrast to vernalization period of donor plants, callus age had no effect on seed number.     

Asymmetric protoplast fusion between sweet potato and its relatives,and plant regeneration

Experiments were conducted to asymmetrically fuse protoplasts from sweet potato (Ipomoea batatas L. Lam.) and its wild relativesI. trifida Don. andI. lacunosa L. Protoplasts of sweet potato were treated with iodoacetamide, whereas those ofI. trifida Don. andI. lacunosa L. were irradiated with X-rays. The asymmetric protoplast fusion was carried out by the electrofusion method and by polyethylene glycol treatment. Electrically-fused protoplasts initiated cell division, and then formed calli earlier than the polyethylene glycol-fused protoplasts. Plant regeneration occurred only in electrofused calli, suggesting that polyethylene glycol had some toxic effect on plant regeneration ability. Analysis of peroxidase isozymes confirmed the interspecific hybrid characteristics of both the fusion-derived calli and regenerated plants.     

稀有植物裸果木的组织培养及植株再生

对稀有植物裸果木进行组织培养研究,在MS培养基上裸果木的下胚轴脱分化形成愈伤组织,并进一步分化形成再生植株。激素种类及其浓度是器官脱分化与植株再生的决定因素。诱导下胚轴形成愈伤组织的最适培养基为MS 1mg／L 6-BA 0．5mg／L NAA;芽分化诱导的最适培养基为MS 1 mg／L6-BA;生根的最适培养基为不含任何激素的1／2MS培养基。     

A general procedure for protoplast recovery,callus and plant regeneration in plants

A portion of this work was supported by NSF grant DCB 8718314 and by the Missouri Agricultural Experiment Station. This research is a joint contribution from the Missouri Agricultural Experiment Station, Journal Series No. 10,789 and from the Massachusetts Agricultural Experiment Station, Amherst, MA 01003; Journal Paper No. MAES 2959. Ousama Zaghmout was supported by the Food for the 21st Century Program, College of Agriculture, University of Missouri, Columbia, MO.     

毛刺槐花药培养及再生植株的获得

以毛刺槐的花药为材料,开展其组织培养和植株再生系统的研究。结果显示：将毛刺槐的花药接种在MS附加2,4—D0．1mg／L和BA3．0mg／L的培养基上,20d时花药愈伤组织诱导率可达41．5％。花药愈伤组织在MS附加BA5．0mg／L的分化培养基上继代培养2个月后,可分化出许多绿色的芽点,待不定芽长至2—3cm高时将其切下,转入MS附加IBA1．0mg／L的生根培养基上,2周后即可得到完整的再生植株。同时,研究就4℃低温预处理和蔗糖浓度对毛刺槐花药培养的影响进行了研究和讨论。     

Plant regeneration in weeping lovegrass, (Eragrostis curvula) through inflorescence culture

Plant regeneration from four genotypes of weeping lovegrass (Eragrostis curvula (Schrad.) Nees), is reported via three developmental pathways: embryogenesis, organogenesis and direct regeneration. Organogenic and embryogenic callus cultures were initiated from young inflorescence segments on Murashige and Skoog's medium supplemented with 2,4-d and BA at different concentrations. The most suitable concentrations of 2,4-d for callus growth and development were 9 and 18 M combined with a BA concentration of 0.044 M. Genotypical differences were observed in the morphogenetic capacity. Direct regeneration was observed under similar culture conditions (culture medium, temperature and photoperiod) but with high light intensity (66 mol m^-2 s^-1). Young plants were successfully transplanted to pots and grown to maturity in the greenhouse.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid