Regeneration capacity of selected genotypes of white mustard (Sinapis alba L.)

Traditional breeding methods based on inbreeding are difficult to implement in the case of Sinapis alba (white mustard) because this plant displays high levels of self-incompatibility. More rapid progress in breeding could be possible if biotechnological methods and in vitro cultures were used. However, white mustard is not readily amenable to biotechnological treatment. Seeds of traditional S. alba cultivars (e.g., Nakielska) are characterized by high levels of glucosinolates and erucic acid. However, a new Polish variety of white mustard (Bamberka) possesses low erucic acid content in the oil. The main goal of the study was elaboration of a plant regeneration system via in vitro culture of hypocotyl and cotyledon explants from low and high erucic acid-containing white mustard cultivars. In these experiments, a simple system for in vitro regeneration of white mustard was developed, with the aim to promote maximum formation of shoots within a short period of time. Traditional and improved cultivars of S. alba showed comparable capacity for shoot development from hypocotyl-derived and cotyledon-derived explants. The two types of cultivars were characterized by essentially equivalent shoot regeneration responses, being slightly higher in hypocotyl than the cotyledonary explants. A greater influence on shoot regeneration from hypocotyl explants was observed on medium supplemented with 4.4 μmol 6-benzylaminopurine, 0.57 μmol indole-3-acetic acid, and a low concentration of kinetin (4.6 μmol). This technique will allow for rapid generation of sufficient plant material for further use in a variety of white mustard breeding projects.     

白芥自交亲和性分析

对不同来源的8份白芥材料,采用人工自交法分析其自交亲和性。结果显示:白芥的自交亲和性存在较大幅度的变异,自交亲和指数在0.01~4.10之间,8份参试材料中,自交亲和指数小于1的材料有3个,自交亲和指数大于1的材料有5个。表明白芥中存在自交亲和材料,白芥自交亲和性变异不仅存在于材料间,而且也存在于同一材料内不同个体间。按自交亲和指数的高低,可将参试材料分为3种类型:高自交亲和类型(自交亲和指数大于3.00,如民乐洪水芥麻、04(X)等)、自交亲和类型(自交亲和指数为1.00~2.99)、自交不亲和类型(自交亲和指数0.00~1.00)。     

Phytochrome-regulated Expansion of Mustard (Sinapis alba L.) Cotyledons

In developing mustard (Sinapis alba L.) seedlings continuousred light acting via the agency of phytochrome stimulates therate of expansion of cotyledons. Although phytochrome actionon cotyledon expansion is evident only after 36 h from sowing,the photoresponse escapes from reversibility at about 15 h fromsowing. The time lag of 21 h between loss of photoreversibilityand the onset of photoregulated cotyledon expansion indicatesthe existence of long-lived components in the phytochrome-triggeredsignal chain. Phytochrome-regulated cotyledon expansion doesnot require the involvement of photosynthesis, as applicationof SAN 9789, an inhibitor of chloroplast biogenesis, did notaffect cotyledon expansion. The role of turgor pressure-relatedcellular parameters such as osmotic potential () cell wall extensibility(m), hydraulic conductivity (L) and yield threshold (Y) forcell expansion were examined during photoregulated cotyledonexpansion. Using the general equation of cell growth dv/dt =[Lm/(L+m)]( - Y), where dv/dt is the rate of volumetric growth,it was demonstrated that the light-mediated cotyledon expansionresults from an increase in cell wall extensibility (m). Theseresults are discussed in relation to the photoregulation ofcotyledon expansion. Key words: Cell wall extensibility, growth, Sinapis alba L., phytochrome     

Die Wirkung von Phytochrom und Actinomycin D auf die Chlorophyll a-Synthese von Senfkeimlingen (Sinapis alba L.)

Zusammenfassung Eine Vorbestrahlung der etiolierten Senfkeimlinge mit 4 Std Dunkelrot bewirkt eine Steigerung der Chlorophyll a-Synthese im Weißlicht. — Andererseits wird die Chlorophyll a-Synthese bereits durch relativ niedrige Actinomycin D-Konzentrationen gehemmt oder verzögert. — Die hemmende Wirkung von Actinomycin D ist — ausgedrückt in Prozent Hemmung — mit und ohne Vorbelichtung genau dieselbe. Auch die übrigen Daten deuten darauf hin, daß Actinomycin D nicht auf die Protochlorophyllsynthese als solche wirkt, sondern vielmehr die Synthese bestimmter Strukturproteine in den Plastiden beeinträchtigt. —Die Daten der Arbeit werden genphysiologisch gedeutet. Der wesentliche Punkt dabei ist, daß aktive Gene (z.B. jene, welche die Enzyme der bekanntlich auch im Dunkeln ablaufenden Protochlorophyllsynthese codieren) relativ unempfindlich gegenüber Actinomycin D sind; potentiell aktive Gene hingegen (z.B. jene, die einige nur im Licht entstehende spezifische Strukturproteine der Plastiden codieren) scheinen sehr viel empfindlicher gegenüber Actinomycin D zu sein. Diese Schlüsse stehen im Einklang mit einer früher geäußerten Hypothese (Lange und Mohr, 1965) und mit den Daten von Schopfer (1966) über die Regulation der Ascorbatsynthese im Senfkeimling durch Phytochrom und Actinomycin D.

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The action of phytochrome and actinomycin D on chlorophyll a formation in mustard seedlings (Sinapis alba L.)

Summary In the mustard seedling chlorophyll a synthesis under white light is enhanced by a pretreatment with far-red which maintains a low but virtually stationary concentration of active phytochrome (=P₇₃₀) (Fig. 1) during the period of irradiation (4 hours). — On the other hand chlorophyll a synthesis is inhibited or delayed by relatively low concentrations of Actinomycin D(=Act) (Fig. 2)The inhibitory action of Act (on a percent basis) is exactly the same with and without a far-red pre-irradiation (Fig. 3). Act in relatively low doses (5 or 10 g/ml) greatly extends the lag-phase of chlorophyll synthesis; however, these doses do not influence the effect of the far-red pretreatment on the rate of chlorophyll synthesis when it finally takes place (Fig.4,5,6). The data presented in this paper indicate that Act does not inhibit protochlorophyll synthesis as such; we have rather to conclude that Act inhibits the de novo synthesis of some specific structural proteins which are prerequisites of chlorophyll accumulation and maintenance in the plastids (Table 1). Synthesis of these structural proteins seems to be under the control of phytochrome too.It is concluded that those genes which are already in function are relatively resistant to Act (e. g. those genes which are needed for protochlorophyll synthesis) whereas potentially active genes (e. g. those which code some specific structural proteins of the plastids) are very sensitive to Act. —A similar conclusion has been reached in an earlier paper in connection with phytochrome-induced antocyanin synthesis (Lange and Mohr, 1965). Our argumentation is further supported by Schopfer's data on control of ascorbate synthesis in the mustard seedling by phytochrome and Act (Schopfer, 1966).

     

Der DNS-Gehalt von Kotyledonen und Hypokotyl des Senfkeimlings (Sinapis alba L.) bei der phytochromgesteuerten Photomorphogenese

Zusammenfassung Der DNS-Gehalt und somit auch die Zellzahl von Kotyledonen und Hypokotyl des Senfkeimlings sind zwischen 36 und 60 Std nach Aussaat sowohl im Dunkel als auch im Dunkelrot weitgehend konstant. Die durch Phytochrom bewirkte Zunahme des RNS-und Proteingehaltes (Weidner u. Mitarb., 1965) muß deshalb als eine RNS-bzw. ProteinZunahme pro Zelle aufgefaßt werden. Dieser Befund stützt die Vorstellung einer differentiellen Genaktivierung durch P₇₃₀ (z.B. Mohr, 1966).—Die weitgehend konstante Zellzahl von Hypokotyl und Kotyledonen während des von uns verwendeten Experimentierzeitraumes ist eine wichtige Voraussetzung für die Vergleichbarkeit biochemischer Daten, z.B. bei kinetischen Studien (vgl. Mohr, 1966).

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The DNA contents of cotyledons and hypocotyl of the mustard seedling (Sinapis alba L.) during phytochrome-mediated photomorphogenesis

Summary DNA contents and accordingly cell numbers of cotyledons and hypocotyl of the mustard seedling were virtually constant during the experimental period (between 36–60 hours after sowing) in the dark as well as under the influence of P₇₃₀, the active phytochrome (Table).—Therefore the phytochromemediated increase of the RNA and protein contents (Weidner et al., 1965) must be understood as an increase of RNA and protein per cell. This fact is in agreement with the conception of differential gene activation mediated by P₇₃₀ (Mohr, 1966). The virtually constant DNA contents during the period of time which is regularly used for experimentation on photomorphogenesis in our laboratory (36–60 hours after sowing; Mohr, 1966) is an important prerequisite for comparing biochemical data under the point of view of differential gene activation, e.g. in kinetical studies in the dark and under continuous far-red light.

     

Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

Die Zellwandzusammensetzung von Hypokotylen des Senfkeimlings (Sinapis alba L.) bei Wachstumshemmung durch Inhibitoren oder Phytochrom

Summary During inhibition of hypocotyl elongation in the mustard seedling by actinomycin D, hydroxyproline, and continuous far-red via phytochrome 730 the synthesis of cell-wall carbohydrates is impaired in the same manner. Independent of the treatment 12 hrs after its beginning at a relative inhibition of hypocotyl elongation of 80–90% the relative inhibition of the cell-wall carbohydrate content is uniformly about 30–35%. No significant quantitative nor apparent qualitative differences in the sugar and uronic acid content of the pectic, hemicellulosic, and -cellulosic cell-wall fraction could be detected.     

Untersuchungen zur Energetik der phytochrominduzierten Photomorphogenese des Senfkeimlings (Sinapis alba L.)

Zusammenfassung Im Zusammenhang mit der Energetik der Photomorphogenese wurden Trockenmasse, Fettgehalt und Verbrennungswärme zu Beginn und Ende des Versuchszeitraums bestimmt und die Kinetik der Sauerstoffaufnahme und Kohlendioxidabgabe aufgenommen. DR-Dauerbestrahlung verschiebt das Maximum der O₂-Aufnahme und CO₂-Abgabe auf einen späteren Zeitpunkt und führt insgesamt zu einer Steigerung des Gasstoffwechsels. Fettabbau und Energieabgabe werden ebenfalls gesteigert, obwohl die Syntheseleistungen des Keimlings (gemessen als fettfreie Trockenmasse bzw. deren Verbrennungswärme) geringer sind als bei den Dunkelkontrollen. Für das Absinken der Ausbeute während der Photomorphogenese werden verschiedene Erklärungsmöglichkeiten angegeben.Eine theoretische Ableitung zeigt, daß der relative Meßfehler bei der indirekten manometrischen Methode gegenüber der direkten Methode mit abnehmendem respiratorischen Quotientem immer größer wird.

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Investigations on the energetics of phytochrome mediated photomorphogenesis in mustard seedlings (Sinapis alba L.)

Summary To characterise the energetics of whole seedlings of Sinapis alba L. during photomorphogenesis under continuous far-red light, measurements were made (between 36 and 72 hours after sowing) of changes in dry weight, fat content, heat of combustion, O₂ uptake and CO₂ evolution in irradiated and dark treated control plants. Irradiation caused a delay in the time of maximum O₂ uptake and CO₂ evolution and led to an increase in the total gaseous metabolism. Likewise, the rates of breakdown of fats and release of energy were increased; this occurred even though the total synthesising capacity of the seedlings, as measured by fat free dry weight or by heat of combustion of fat free dry matter, was lower in the farred irradiated seedlings than in the dark controls. Thus, during the course of photomorphogenesis the yield of plant material, as measured by the increase in fat free dry matter divided by the decrease in fat, or by the increase in heat of combustion of fat free dry matter divided by the decrease in heat of combustion of fat, was markedly reduced (approximately 60%). Several possible explanations were put forward to account for this reduction, but lack of thermodynamic data allowed none to be favoured.Theoretical considerations showed that the experimental error associated with measurements of gaseous metabolism was always larger when the indirect manometric method rather than the direct method was used. This effect was more pronounced the higher the respiratory quotient was.

     

Isolation and Characterization of Mustard (Sinapis alba L.) Seed Storage Proteins

A total storage protein fraction was prepared from mustard (Sinapis alba L.) seeds via isolated protein bodies and characterized by sedimentation, immunological, and electrophoretic techniques. Mustard seed storage protein consists of three fractions (1) a “legumin-like” 13-S complex composed of two pairs of disulfide-linked polypeptides (16.5 + 28.5 kDa and 19.5 + 34 kDa, respectively) and two single polypeptides (18 kDa and 26 kDa), (2) a “vicilin-like” 9-S complex composed of two glycoproteins (64 kDa and 77 kDa), and (3) two small polypeptides (10 kDa and 11 kDa) which probably represent the 1.7-S complex found in other Cruciferae. In contrast to related species, no glycosylated polypeptide was found in the 13-S complex. Immunological relationships were found between the paired polypeptides of the 13-S complex but not between polypeptides of the 13-S complex and polypeptides of the 9-S complex. Pulse-chase labeling and in vitro translation of polysomal RNA from young embryos demonstrated that the polypeptides of the 13-S complex originate from high molecular mass precursors, except for the 18 kDa polypeptide which appears to be synthesized in its final size. The amino-acid composition of the major polypeptides of the mustard storage protein is given.     

Interspecific somatic hybridization between Brassica napus and Brassica hirta (Sinapis alba L.)

Summary Somatic hybridization between Brassica napus and B. hirta (or Sinapis alba) is described. No cybrid plant with B. napus nucleus exhibiting cytoplasmic male sterility was recovered. Somatic hybrids were identified morphologically and, for some of them, by cytological observations. They were also characterised by Southern hybridization of nuclear rDNA. Chloroplast and mitochondrial DNA restriction analysis showed that 2 plants out of 14 have B. hirta ctDNA, one the B. napus mtDNA and the other a hybrid. Nine possess B. napus ctDNA with a hybrid mtDNA. For six of them, mtDNA patterns present novel bands, suggesting intergenomic recombination during fusion. These hybrids will be included in the breeding program.     

Analysis of light-controlled accumulation of carotenoids in mustard (Sinapis alba L.) seedlings

Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR high irradiance reaction (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

Myrosinase in Sinapis alba L.

Extracts from developing seeds, seedings, and different organsof mature plants of Sinapis alba L. were tested for the presenceof myrosinase. Three different patterns of isoenzymes were distinguishedby isoelectric focusing. One was found in developing seeds andseedlings, a second in mature leaves, stems, flowers, and podsand a third in roots. Sections of all the parts tested were examined for the presenceof myrosin cells by light microscopy. Developing seeds and seedlingsshowed a good correlation between the appearance of myrosinaseand the presence of myrosin cells. No myrosin cells were foundin root or flower parts although they showed myrosinase activity.