In vitro clonal propagation of annatto (Bixa oreliana L.)

Summary A protocol for in vitro propagation of Bixa orellana is described. Plants were regenerated from shoot apex and nodal explants on B5 medium supplemented with 4.9 μM 2-isopentenyl adenine. The multiplication factor of shoot apex explants was higher (nine shoots per explant) than that of the nodal explants (five shoots per explant). Regardless of the position of the nodes, all the nodal explants gave similar responses. However, the size of the nodal explant was an important factor in producing multiple shoots: 0.5 cm nodal explants produced the maximum multiple shoots. Regenerated shoots from shoot apex explants rooted best on MS medium supplemented with 0.05 μM α-naphthalene acetic acid (NAA). whereas shoots regenerated from nodal explants needed 2.7 μM NAA for rooting. Eighty per cent survival of in vivo transferred plants occurred on the best potting substrate, coco peat. Since the multiplication factor was nine per explant, this protocol can be use for commercial microprogation. However, the regeneration capacity declined after 10 subcultures. Approximately, 3350 rooted plants could be generated in 10 mo. after eight subcultures, from one shoot with a shoot apex and four nodes.     

Callus induction and plantlet regeneration in ornamental Alocasia micholitziana

Callus induction from petiole explants has been achieved in Alocasia micholitziana `Green Velvet'. The highest percentage (71%) of explants inducing callus was obtained on MS medium supplemented with 0.5 M 2,4-D and 0.5 M kinetin in the dark after 4 months of culture. Shoots were regenerated at the highest frequency of 33.3% under light condition when 0.5 M BA was added to MS medium with the average of 7.8±2.3 shoots per callus explant. The callus-derived shoots rooted on hormone free MS medium and within 4 weeks the plantlets were ready for acclimatization. The regenerated plants appeared morphologically similar to mother plants.     

Callus induction and plantlet regeneration in damiana (Turnera diffusa,Willd.)

Experiments were performed to determine optimal conditions for callus induction and plantlet regeneration from damiana (Turnera diffusa, Willd.) leaf explants. MS media [7] and B5 media [4] were used; both of them were supplemented with different combinations of 2,4-dichlorophenoxyacetic, acid (2,4-D) and 6-benzyl-adenine (BA). In all cases the samples were incubated under continous fluorescent light, 100 mol.m^-2.sec^-1, at 25±2°C for 60 days. Highest callus percentage was obtained in MS media supplemented with 2,4-D 10^–5 M plus BA 10^-8M. Maximum percentage and multiple shoots and leaves were produced in MS media with BA 10^-5M and BA 10^-5M plus 2,4-D 10^-7M. These explants were transferred for rooting to MS medium without plant growth regulators. Later, rooted plantlets were successfully established in soil.     

Callus induction and plantlet regeneration inCichorium intybus L.: II. Effect of different hormonal treatments

Summary Expiants ofCichorium intybus L. storage roots were grownin vitro on a modified Heller's medium lacking auxins and cytokinins, or supplemented with auxins (either 2,4-D or NAA) alone or with a cytokinin (kinetin) or auxin and kinetin combinations in different concentrations. The morphogenetic responses of root explants varied with the different hormonal treatments. The best response for callus growth was obtained in presence of 2,4-D. On the contrary, kinetin alone was very effective for shoot induction, increasing the formation of adventitious buds (up to 100% of the explants) in respect to control (hormone-free medium). NAA induced either shoot differentiation (in a medium frequency) or root formation. Expiants excised from root zones near to apex, which showed on hormone-free medium a very low regenerative capacity (lower than proximal zones of the root), responded to kinetin by increasing significantly the number of shoots from adventitious buds.Cytological analyses in developing primary calli showed, in all media, high incidence of amitotic phenomena confirmed by DNA cytophotometry in calli at different growth stages. The histological analysis demonstrated the formation of meristematic growth centers on the organogenesis inducing media and the subsequent development of these meristemoids as shoot (or root) apices in the callus mass.The results are discussed in comparison with previous observations of the authors inCichorium intybus (Caffaro et al. 1982) and in relation to the action of hormonal treatments on callus formation and organogenesis. The cytological and histological results are also discussed in relation to the hormonal composition of the medium.     

Callus induction and plantlet regeneration from mature cotyledonary segments of groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

We evaluated the efficiency of callus induction and plantlet regeneration from mature cotyledonary segments of groundnut cultivars VRI-2 and VRI-3. Callus cultures were induced from mature tissues using NAA and IAA in combination with KIN or BAP. Maximum induction was recorded with 3.0 mg/L IAA and 1.0 mg/L BAP. However, green, compact, and nodular calli were obtained in 2.5 mg/L of IAA or NAA combined with 1.0 mg/L of either BAP or KIN. Fresh and dry weights were highly influenced by auxin concentration. Compact and nodular calli were then transferred to shoot induction media. The highest mean number of shoots was observed in 3.0 mg/L BAP plus 0.5 mg/L IAA. Finally, the resulting plantlets were rooted with IBA and NAA.     

海甘蓝愈伤组织再生植株的研究

海甘蓝种子在附加有２～５ｍｇ／Ｌ６－ＢＡ＋０１ｍｇ／ＬＮＡＡ的ＭＳ培养基上,幼苗生长健壮。幼苗的下胚轴和子叶柄在ＭＳ＋１ｍｇ／Ｌ２,４－Ｄ＋０５ｍｇ／Ｌ６－ＢＡ的培养基上可以获得较好的愈伤组织。将来源于下胚轴的愈伤组织培养于含有０５ｍｇ／ＬＮＡＡ,２ｍｇ／Ｌ６－ＢＡ的ＭＳ培养基上分化出的丛生芽状态最好。最佳生根培养基为１／２ＭＳ＋０５ｍｇ／ＬＢＡ。     

Callus induction and high-frequency plant regeneration from hypocotyl and cotyledon explants of Arctium lappa L.

Summary This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l^&#x2212;1 2,4-dichlorophenoxyacetic acid and 0.5&#x2013;2.0 mg l^&#x2212;1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0&#x2013;2.0 mg l^&#x2212;1 &#x03B1;-naphthaleneacetic acid (NAA) and 0.5&#x2013;2.0 mg l^&#x2212;1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^&#x2212;1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^&#x2212;1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

Callus initiation and plant regeneration in ragi (Eleusine coracana Gaertn.)

Callus was successfully initiated on root, mesocotyl and leaf base segments of 3- to 4-day-old seedlings of ragi (Eleusine coracana Gaertn.). 2,4-D along with casein hydrolysate for Murashige and Skoog's basal medium was found to be most effective for callus initiation and maintenance. Mesocotyl and leaf base tissue derived calli gave shoot buds in medium in which the 2,4-D concentration was lowered.     

Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast

The tissue culture of phycocolloid yielding seaweeds included preparation of axenic explants, callus induction, subculture of excised callus and regeneration of plantlets from pigmented callus in the laboratory. Treatment of algal material with 0.1–0.5% detergent for 10 min and 1–2% betadine for 1–5 min and 3–5% antibiotic treatment for 48–72 h successively enabled viable axenic explants to be obtained as high as 60% for Gracilaria corticata, Sargassum tenerrimum and Turbinaria conoides and 10% for Hypnea musciformis. Callus induction was more conspicuous in T. conoides than in the other three species investigated. Of the irradiances investigated, 30 μmol photons m⁻² s⁻¹ produced calluses in as many as 40% explants in G. corticata and T. conoides and 10% in H. musciformis and S. tenerrimum. The explants cultured at 5 and 70 μmol photons m⁻² s⁻¹ did not produce any callus in all the species studied except for H. musciformis in which 10% explants developed callus at 5 μmol photons m⁻² s⁻¹. Most of the species investigated showed uniseriate filamentous Type of growths and buds from cut ends and from all over the surface of explants. Nevertheless, T. conoides had three Types of callus developments, namely (1) uniseriate filamentous Type of outgrowths from the centre of the cut end of explant, (2) bubbly Type of callus and (3) club-shaped callus clumps. The subculture of T. conoides callus embedded in 0.4% agar produced two Types of filamentous growth, namely filiform (with elongated cells) and moniliform filaments (with round cells) in the 2 months period after inoculation. Further, friable callus with loose cells was also found associated with excised callus. The moniliform filaments showed prolific growth of micro-colonies resembling to somatic embryo-like growth which, in liquid cultures, differentiated and developed into propagules with deformed shoots and distinct rhizoids. The shoots of these propagules remained stunted with abnormal leaf stalks without forming triangular shaped leaves as the parental plant and rhizoids had prolific growth in the laboratory cultures. The excised callus of G. corticata continued to grow when transferred to liquid cultures and showed differentiation of new shoots within 10 days. The shoots grew to a maximum length of 5–6 cm in the 2 months period in aerated cultures in the laboratory. Dedicated to the memory of Late Dr. Rangarajan.     

Callus growth and plant regeneration in diverse cultivars of cucumber (Cucumis sativus L.)

The growth and differentiation of callus tissues derived from cotyledons of ten cultivars ofCucumis sativus L. were investigated. Cotyledonary explants from all ten cultivars formed callus tissue on Murashige and Skoog (MS) medium supplemented with 0.5 M 2,4-dichlorophenoxyacetic acid and 5 M 6-benzylaminopurine. Fresh weight of the callus tissues averaged 1 to 8 g per flask after five weeks of culture. Shoot development was achieved in three cultivars, Hukchinju, Manchoonchoungjang and Seoul, on MS medium supplemented with 0.5 M -naphthaleneacetic acid and 5 M 6-benzylaminopurine. Reducing the 6-benzylaminopurine concentration to 0.01 M resulted in root formation on callus tissues and on shoots transferred to this medium. All cultivars gave the same response in tests of root formation, but shoot regeneration from callus culture of cucumber cotyledons was dependent on genotype with cultivar Manchoonchoungjang exhibiting the best shoot differentiation capability among the genotypes examined. Examination of mitotic metaphase from the regenerants revealed that all were tetraploid.     

不同红掌品种的叶片、叶柄和茎段愈伤组织的诱导及植株再生

红掌幼嫩叶片表面灭菌的最佳方法是：0．01％KMnO4 10min,0．05％链霉素、0．05％制菌霉素和0．05％头孢唑林钠各灭菌20min,然后用75％乙醇30s和HgCl2 2min,污染率为零。Anthurrium adraeanum “Rosetta“和A.adraeanum “Oilcloth-flower”愈伤组织诱导率和芽分化率显著高于A.adraeanum “Atlanta”.A.adraeanum “Fantasis”,A.adraeanum“Afrikanerin”。不同品种茎段、叶柄、叶片的愈伤组织诱导率和芽分化率有明显差异,大多数品种是幼茎段＞叶柄＞叶片。幼苗移栽在草煤或血竭渣基质中,成活率可达97％。     

Callus initiation and regeneration capacities in Brassica species

In order to know the genetic differences of de- and redifferentiation capacities, seven Brassica species (B. campestris, B. nigra, B. oleracea, B. hirta, B. carinata, B. juncea and B. napus) were cultured in vitro, and their response to the medium supplemented with various combinations of auxin and cytokinin hormones was compared. Important factors for callus initiation were shown to be auxin and species. Calli were induced most frequently in Murashige and Skoog (MS) medium with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), whereas -naphthaleneacetic acid (NAA) induced preferentially roots at a concentration of 2 to 5 mg/l. Callus-, root- and shoot-forming capacities from explanted cotyledon tissues were significantly different among the seven Brassica species. Calli derived from cotyledons and hypocotyls of seven species were transferred to MS media with 20g/l sucrose, 0 to 0.1 mg/l NAA and 0 to 4 mg/l kinetin to compare their regeneration capacities. Among the seven species tested, B. napus (2n=4x=38, genome AACC) had the highest shoot forming capacity (20.0%). Other amphiploid species, B. carinata (2n=4x=34, BBCC) and B. juncea (2n=4x=36, AABB) formed shoots at low frequencies (2.8% and 1.2%, respectively). A diploid species, B. oleracea (2n=2x 18, CC) also showed high shoot formation (10.2% on average). This result suggests that the gene(s) controlling shoot formation may be localized in the C genome. Differences were also found among varieties and cultivars within a species. One of the cultivars, Siberian kale (B. oleracea var. acephala) gave about 50% shoot formation. This kale was shown cytologically to be an autotetraploid (2n=4x=36, CCCC), thus probably possessing a double set of the shoot-forming gene(s).     

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     

Callus culture of Coronilla varia L. (crownvetch): plant regeneration through somatic embryogenesis

Callus culture and plant regeneration through somatic embryogenesis have been obtained in Coronilla varia. Media used were UM (25) supplemented with 2 mg/l 2,4-D followed by subculture on MS (18) containing 1 mg/l 2-iP and 0.1 mg/l IAA. Embryoids developed into complete plantlets on filter paper saturated with hormone-free MS medium.     

反向核不育水稻FHS组织培养高频率植株再生

以反向核不育水稻品系FHS为研究材料,对其成熟胚愈伤组织的诱导及其再生条件进行了研究。研究结果表明,通过选择合适的培养基和调节2,4-D的浓度,胚性愈伤组织的诱导率可以达到92.5%。通过调节MS培养基中KT和NAA的浓度,并加入适量的CuSO4,植株再生频率高达81.3%。由此可见,合适的激素浓度的高低与FHS植株再生频率的高低呈现出一定的相关性。     

The effect of carbon source on callus induction and regeneration ability in Pharbitis nil

Flower buds, cotyledons and hypocotyls of Pharbitis nil were used as plant material. Flower buds (1–2 mm long) were excised from 3-week-old plants, grown in soil. Cotyledons of 7-day-old sterile seedlings were cut into 25 mm² squares cotyledons whereas hypocotyls were cut to 1 mm long fragments. Explants were transferred into Petri dishes containing the Murashige and Skoog medium (MS), supplemented with either BA (11 μM·L⁻¹) alone or BA (22 μM·L⁻¹) and NAA (0.55 μM·L⁻¹), and different sugars: sucrose, fructose, glucose, mannose or sorbitol (autoclaved or filter-sterilized). Addition of glucose instead of sucrose to the medium stimulated the induction of callus on flower buds and cotyledonary explants, but inhibited its growth on fragments of hypocotyls. The medium supplemented with fructose (especially filter-sterilized) stimulated the development of flower elements. Organogenesis of shoots and roots on explants was also observed. Flower buds and hypocotyls were able to regenerate both organs. Addition of fructose or glucose to the medium stimulated the organogenesis of shoots, whereas root organogenesis was inhibited on all explants used. Sorbitol strongly inhibited both induction of callus and organogenesis on all explants used.     

Callus induction and thallus regeneration in some species of red algae

Callus induction was obtained from axenic explants of 14 species of red algae. ASP12NTA solid medium (1.5% agar) supplemented with indole-3-acetic acid (IAA) and 6-benzylaminopurine (BAP) was used for callus induction. In most of the species, addition of IAA or BAP at 0.1 mg/L or 1.0 mg/L enhanced callus induction rate or callus size. The combination of IAA (0.1 mg/L) and BAP (0.1 mg/L) was more effective among eight species, while high concentrations of IAA (10 mg/L) showed an inhibitory effect. Great variation in callus form, source tissue, and color of the induced callus were observed. The callus mainly originated from medullary and cortical tissue of the explant. Callus with filamentous, oval and spherical cell chains or disorganized cell mass was observed. The excised calluses from the explants of six species showed sustained growth on subculture. On transfer of the subdivided callus mass of seven species to PES liquid medium, shoot formation and thallus regeneration were observed.