Djelovanje regulatora rasta na preživljenje fragmenata hipokotila bundeve

Basic Heller’s medium is not suitable for the growth and development of fragments of pumpkin hypocotyls. On this medium the fragments hardly survive a culture period of one month. The addition of growth regulators (IAA, 2,4-D or kinetin) to Heller’s medium delays the senescence of the tissue and its decay. Moreover, it provokes cell divisions resulting in an increase of fresh and dry weight

Razvitak abnormalnih embrioida u embriogenom kalusu bundeve (Cucurbita pepo L.)

In addition to normal forms of embryoids in an embryogenic callus of pumpkin, various abnormal forms also appear. They can be characterized, according to their morphology, as monocotyleous, heterocotyleous, tricotyleous, quadricotyleous, syncotyleous, and embryoids with leaf-like cotyledons. Other forms observed include: twins, forms with undeveloped roots, undeveloped hypocotyls, callus formed on the surface of the plantlet, multiple growth and forms of undifferentiated masses. The anomalies described, with the exception of certain differences in the size of the embryoids are not dependent on the presence of a specific growth substances in the nutrient medium

Rizogeneza na eksplantatima hipokotila bundeve

Hypocotyl fragments of pumpkin were grown on Heller’s synthetic medium or MS-medium with 3°/o glucose and 0.9% Difco Bacto agar and some growth regulators. Heller’s basic medium is not suitable for the growth and development of hypocotyls. On this medium the fragments never show any signs of differentiation and hardly survive the culture period of one month. Moroever, as shown earlier, IAA with kinetin or 2,4-D with kinetin in Heller’s medium provoke cell divisions resulting in an increase of fresh and dry weight but never in rooting. In a small number of expiants with only 2,4-D at the concentrations of 10-7 and 10-6 very weak rhizogenesis was induced. The MS-medium has proved to be much more suitable. The medium consisting of MS-salts and sugar only is sufficient for root iduction on the root side of the fragment; for the vigorous root growth, however, the complete MS-medium is necessary. The addition of some substances, e. g. of autoclaved water-melon sap (15% vol.) or autoclaved water-melon sap combined with 2,4-D (3.10-7) further intensifies the vigorous growth of the roots; low concentrations of NAA, high concentrations of IBA and IAA (10-6) with kinetin (3.10-7) show the same effect.Fragmenti hipokotila bundeve veličine 1 cm bili su uzgajani na osnovnom Hellerovom mediju ili MS-mediju uz dodatak 3% glukoze, 0,9% Difco Bacto agara i različitih regulatora rasta. Osnovni Hellerov medij nije bio pogodan za rast i razvitak eksplan- tata hipokotila bundeve. Na ovom osnovnom mediju, bez regulatora rasta, fragmenti nisu pokazivali nikakvu diferencijaciju, pa ni rizogenezu, i nisu preživjeli period kultiviranja od mjesec dana. Dodatak 2,4-D, IAA i kinetina produžio je preživljenje eksplantata preko mjesec dana, no niti na j različiti je kombinacije 2,4-D i kinetina, a tako ni IAA i kinetina, nikada nisu potakle stvaranje korijena. Jedino je na manjem broju kultura uočena pojava slabih korjenčića nakon dodatka same 2,4-D u visokim koncentracijama. MS-medij bio je mnogo pogodniji. Sama podloga MS-soli uz dodatak 3% glukoze bila je dovoljna da se korijenje inducira na proksimalnoj (korijenskoj) strani eksplantata. Bujno korijenje naraslo je međutim na kompletnom MS-mediju. Bez dodatka šećera na kompletnom MS-mediju nije bilo moguće izazvati rizogenezu. Dodatkom izvjesnih supstancija rasta pojačao se broj induciranog korijenja i njegov razvoj. Tako je npr. dodatak od 15% soka lubenice osnovnom mediju MS-soli djelovao poput dodatka aminokiselina i vitamina iz kompletnog MS-medija. Dodavanjem 2,4-D u koncentracijama 10 na-6 i 10 na-7 te NAA u nižim koncentracijama (10 na-9 i 10 na -8), kombinacija IAA 10 na -6) i kinetina (3.10 na-7) te IBA u visokim koncentracijama (10 na-6) kompletnom MS-mediju postignut je bujni rast korijenja na eksplantatima hipokotila bundeve

Influence of meristem culture and virus elimination on phenotypical modifications of grapevine (Vitis vinifera L., cv.Refošk)

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Utjecaj L-prolina na somatsku embriogenezu u dugotrajnim kalusnim kulturama ječma (Hordeum vulgare L.)

Long-term callus cultures of barley (Hordeum vulgare L.) with decreased embryogenic potential were used in testing the influence of L-proline on somatic embryogenesis. The addition of up to 10 mM L-proline to the induction medium increased both the frequency of embryoid formation and the number of subsequently regenerated plantlets. Most of the plantlets (82%), however, were albinos. Some callus lines retained embryoid and plantlet formation capacity after being in culture for more than 21 months.Dugotrajne kalusne kulture ječma (Hordeum vulgare L.) sa smanjenim embriogenim potencijalom korištene su za ispitivanje utjecaja L- prolina na somatsku embriogenezu. Dodatak do 10 mM L-prolina u indukcijsku podlogu povećao je ne samo učestalost nastanjanja embrioida nego i broj biljčica koje su naknadno regenerirane. Ipak, većina biljčica (82%) bila je albino. Neke kalusne linije zadržale su sposobnost stvaranja embrioida i biljčica i nakon što su kultivirane više od 21 mjesec

Regeneracija biljčica u kulturi vegetacijskog vrška vrste Pelargonium zonale hybrid

Shoot tips (0.5 mm high) of Pelargonium zonale hybrid cvs. ’Career’ and ’Fire flash’ were cultured on modified MS medium: macroelements (Murashige and Skoog 1962), microelements (Nitsch and Nitsch 1970) containing (mg 1^1) thiamine.HCl 0.4, m-inositol 200, caseine hydrolysate 1000, IAA 0.5, kinetin 10.0; (g 1-1) sucrose 30, bacto agar 6; pH 5.8. The ’Career’ did not grow on this medium, while the ’Fire flash’ did: 30% of shoots produced an organogenic callus, which was capable of being continuously subcultured. The shoots and plantlets obtained rooted well on a medium containing 0.1 mg 1-1 IAA only, and after a month they were transferred into pots. Callus cultures and in vitro formed plants, examined for Xanthomonas pelargonii and other bacteria, were found to be free of pathogens.Vegetacijske vrškove (veličine 0,5 mm) biljke Pelargonium zonale hybrid 2 sorte: ’Career’ i ’Fire flash’ kultivirali smo na modificiranom MS mediju (Makroelementi po Murashige i Skoogu 1862, mikro- elementi po Nitsch i Nitschu 1970) uz dodane slijedeće tvari: (mg 1-1) tiamina.HCl 0,4; m-inositola 200; hidrolizata kazeina 1000; 0,5 kinetina 10,0 te (g 1-1) saharoze 30 i Difco bakto-agara 6; pH 5,8. Dok sorta ’Career’ nije rasla na mediju ovog sastava, sorta ’Fire flash’ \u27e na 30% eksplantata proizvela dobro rastući organogeni kalus koji se mogao supkultivirati. Izdanci i biljčice regenerirani in vitro dobro su se zakorjenjivali na osnovnom mediju uz dodatak samo 0,1 mg 1-1 IAA i u velikom postotku preživljavali presađivanje u lonce. Testiranje kalusnih kultura i in vitro regeneriranih biljaka na prisutnost bakterija Xanthomonas pelargonii i ostalih bakterija dalo je negativne rezultate

The Effect of 2,4-D and 2-NOXA on the Increase of the Number of Cells in Explants of Pumpkin

By the method of Steward and Shantz (1955) it has been attempted to determine the effect of 2,4-D and 2-NOXA (cone. 10~9 — 10~ 6) on the increase of the number of cells in explants of pumpkin. The cells were counted at the begining of the experiments on a sample (0), which was very similar to the explants under treatment and at the end of the experiments which lasted 25 days. For comparison countings on explants cultivated on the medium without addition of growth-stimulators were used. The results obtained are shown in graphs by means of simple columns, 1 mm column height corresponding to 100,000 cells. It is evident that the cell number of explants, which grew on a medium with 2,4-D and 2-NOXA at higher concentrations (1Q~7 — 10~5) was larger in comparison to the control as well as to the explants grown at lower concentrations of the substances investigated

Growth of Fragments of mature Pumpkin Embryos cultivated in vitro

The growth of the segments A and B of mature embryos of pumpkin (Cucurbita pepo L.) has been studied in vitro. The segments were 0,5—0,75 mm long. The segment A corresponded to the upper part of hypocotyl tissue with epicotyl and apical meristem, the segment B to the lower part of the hypocotyl, which was deprived of correlative relations. The experiments have given the following results: When submerged, the embryo segments did not show any signs of organogenesis and the increase of fresh and dry weight was considerably smaller than in segments which were cultivated on the surface of the medium. From three different nutrient media, which were used, (Heller’s Rob- bin’s and White’s) Heller’s medium was the most suitable one and caused the highest increase in fresh and dry weight of explants. However, the segment A was able to grow into a normal plant only on Murashige—Skoog medium. These facts are quite in agreement with our further investigations on the needs of pumpkin tissue for a basic medium rich in salts e. g. the MS-medium. When cultivated on the surface of a basic nutrient medium, say Heller’s medium, the segments differentiated into hypocotyls (the A- segments became 4—5 cm long, and the B-segments 0,5—1 cm) and into weak roots (on the proximal side of segments). The application of IAA in high concentrations (10-5) induced roots also on the distal part of the segments, especially on B-segments. As a rule the roots were more vigorously developed in B-segments. The addition of coconut milk in concentration of 10% stimulated the development of the hypocotyl, just as the addition of the IAA in a concentration of 10% It is interesting that after a cultivation with adenine the A-segments were more vigorous than in the control. An addition of 0,5% yeast extract to the medium containing coconut milk also allowed a vigorous growth of the hypocotyl. In concentrations 10-6 and 10-5 the IAA increased the number of induced roots, disturbed the polarity of their formation and inhibited their elongation. High concentrations of growth stimulators, as e. g. 2,4-D (10-6 and 1CT5) or 2,4,5-T (10-6 and 10-5) inhibited any organogenesis. The addition of 2,4-D (10-6) to the medium containing coconut milk, prevented the action of coconut milk so that the development of hypocotyl and roots was inhibited as compared to the growth of undifferentiated tissue which was slightlly stimulated. In concentrations of 10-8 and 10% kinetin had a strong inhibitory effect on the development of hypocotyl even without any addition of other substances. At a concentration of 10-7 the kinetin enabled the development of the epicotyl with the apex. The addition of both kinetin and IAA (both in concentration 10-6) strongly inhibited the development of hypocotyl. In addition to the inhibition of the hypocotyl differentiation high concentrations of growth stimulators (e. g. 2,4-D) induced cell dedifferentiation and the appearance of cells of embryonic type, about 20 micro m in diameter