Development of secondary inflorescences and in vitro plantlets from inflorescence cultures of Amaranthus paniculatus

Immature inflorescences of Amaranthus paniculatus were used as explants for in vitro culture studies. When placed on a medium supplemented with 3–6 mg/l kinetin, explants developed into secondary inflorescences. Leaves and shoots developed following culture of inflorescence tissue on media containing 8–15 mg/l kinetin or 5–10 mg/l BAP. These shoots when subcultured on MS medium supplemented with 12 mg/l kinetin + 15% coconut milk, formed roots. These rooted plantlets later flowered in vitro.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - CM coconut milk     

Haploid Plantlet Formation from Female Gametophytes of Ephedra foliata Boiss. in vitro

Female gametophytes (at the archegonial stage) excised fromyoung ovules of Ephedra foliata Boiss, were cultured on a basalmedium (Murnshige and Skoog's combinations of major and minorsalts, Iron source, vitamins, myo-inositol along with 2 percent sucrose and 10 per cent coconut milk) under aseptic conditions.Growth and morphogenetic responses of the explants to auxinswere compared at different concentrations and a study of theirinteractions with cytokinins has also been made. At 2 mg 1^–1,2, 4-D induced profuse callusing which subsequently producedroots. NAA at 4 mg 1^–1 was optimal for callus growth androoting. Combinations of 2,4-D and kinetin were more effectivein inducing roots and shoot buds than those of 2,4-D and benzylamino-purine (BAP). Addition of BAP (0.05 mg ^1–1) to themedium containing optimal concentrations of NAA resulted information of a large number of roots. Kinetin induced only rootingin the presence of 4 mg 1^–1 NAA. A high concentrationof BAP (8 mg 1^–1), stimulated shoot bud formation. Forthe further development of shoot buds, neither auxin nor cytokininwas needed. Cytological observations revealed the presence ofhaploid number of chromosomes, i.e. seven. Ephedra foliata, tissue culture, callus, regeneration, 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid, kinetin, benzyl amino-purine     

In Vitro Culture and Regeneration of Solanum khasianum and Extraction of Solasodine

Leaf explants of Solanum khasianum regenerated on MS medium containing 2, 4-D (3 mg/l) and kinetin (1 mg/l). Shoots could be induced from these calli on medium containing BAP (3 mg/l) alone. Rhizogenesis of these shoots occurred when transferred to medium containing 2 mg/l NAA. The yield of solasodine — a pharmaceutically important compound, from 4-month-old callus tissue was remarkable at 2 per cent of dry weight.     

Morphogenic Potential of Cultured Explants of Crocus chrysanthus Herbert. cv. E. P. Bowles

Explants of leaves, basal plates, petals, anthers and ovariesof young growing corms of Crocus chrysanthus var. E. P. Bowleswere cultured on MS basal media with 20 different combinationsof either kinetin and NAA or BAP and 2, 4-D in the dark. Nomajor change was observed except on ovary explants. The ovaryexplants produced callus at 5.0 mg 1^–1 and 10 mg^–1BAP and subsequently stigma-like structures formed on the surfaceof the callus. Transfer to light resulted in the stigma-likestructures developing a yellow pigmentation whereupon they cameto resemble the naturally-grown stigmas. Corm formation andshoot regeneration was obtained from the callus when the ovaryexplants were cultured on media containing 5.0 and 10 mg I^–1BAP with 0.5 mg 1^–1 2, 4-D. Increasing the level of 2,4-D markedly reduced the number of shoots produced per explant. Key words: Crocus chrysanthus, callus, ovary explants     

Morphogenesis and plant regeneration from cultured endosperm of Emblica officinalis Gaertn

Mature endosperm of Emblica Officinalis (Euphorbiaceae) formed a continously growing callus on MS medium supplemented with an auxin (2,4-D or IAA) and a cytokinin (K or BAP). Subculture of callus on MS with BAP (0.2 mg/l) and IAA (0.1 mg/l) resulted in formation of shoots and embryo-like structures in 50 and 8 per cent cultures, respectively. Regeneration of shoots was more frequent when both BAP (0.2 mg/l) and IAA (0.1 mg/l) were present than on BAP (0.2 mg/l) alone. The embryo-like structures produced plantlets.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indoleacetic acid - K kinetin - NAA naphthaleneacetic acid - PDB para-dichlorobenzene (née Arora)     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

NUTRITIONAL AND MORPHOGENETIC INVESTIGATIONS ON CALLUS CULTURES OF NEOMAMMILLARIA PROFILERA MILLER (CACTACEAE)

Neomammillaria prolifera (Cactaceae), when grown on Murashige and Skoog's medium supplemented with fresh coconut milk, showed very little growth. Various concentrations and combinations of growth regulators which did not cause callusing had no apparent effect on the normal growth rates of intact plants. Healthy green calli obtained on a 2,4-D and kinetin-containing medium exhibited extremely fast growth and very specific growth requirements. Relatively high amounts of 2,4-D (10–20 mg/liter), kinetin (1–2 mg/liter), and coconut milk (20–60%) were required at all times for continued proliferation of callus on subculturing. Moreover, the callus was very tolerant to extremely high concentrations of other growth regulators (IAA, NAA, IBA, and GA up to 100 mg/liter) in the presence of 2,4-D and coconut milk. These substances could not replace 2,4-D for callusing or continued growth of callus. It was not possible to establish root cultures or to induce callusing of roots. Attempts to induce differentiation in callus were unsuccessful, except for sporadic root initiation in some cultures. A comparison of these results with similar studies on other succulents demonstrates some basic physiological similarities among this group of plants.     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

INDUCED DIFFERENTIATION OF GERANIUM PLANTS FROM UNDIFFERENTIATED CALLUS IN VITRO

Geranium callus produced from explants of stem tip, internode pith with vascular tissue on synthetic media with or without coconut milk and 2,4-dichlorophenoxyacetic acid grew well for many generations, but only tracheids were induced in them. If callus produced on these media was subcultured immediately on Murashige and Skoog medium with 0.1 mg/liter/α-naphthalene acetic acid (NAA) and 10.0 mg/liter kinetin (K) and incubated at 16/8-hr light/dark cycle, shoots were induced in 8-10 weeks and roots in another 8-10 weeks. Callus produced on the MS medium with the same supplements of NAA and K, subcultured on the same medium and incubated in 16/8-hr light/dark cycle, produced shoots in 6-8 weeks. However, on callus subcultured more than three times, shoots differentiated with greater difficulty and none differentiated after six subcultures. Some abnormal shoot-like structures were also produced, the cells of which showed virus-like inclusion bodies. Requirements of the different varieties for differentiating organs differed. Among 12/12-, 15/9-, 16/8-, and 20/4-hr light/dark photoperiods that induced differentiation, 15/9- and 16/8-hr were more effective than the others. Continuous illumination did not induce differentiation. Differentiated shoots formed roots more readily on a medium with reversed proportions of auxin and kinetin. On agar roots were devoid of root hairs. Root hairs were formed when the shoots and plantlets were cultured on filter-paper bridges. Many “mother” stock plants were produced. These are being studied for their growth qualities and for possible viruses and other pathogens.     

Chemical Regulation of Callus Growth, Organogenesis, Plant Regeneration, and Somatic Embryogenesis in Antirrhinum majus Tissue and Cell Cultures

Excised stem explants of Antirrhinum majus L. var. ‘Kymosyblanc’ were grown in a denned medium to investigate factorsinfluencing bud and root development, callus induction, andsomatic embryogenesis. Auxins such as indoleacetic acid (IAA)and naphthaleneacetie acid (NAA) caused limited callus developmentand abundant root formation, whereas 2,4-dichlorophenoxyaceticacid (2,4-D) promoted soft friable callus with embryos and occasionaldevelopment of thick abnormal roots. 2-Naphthoxyacetic acid(NOA) and coconut milk (CM) used together induced friable greencallus growth and differentiation of small globular embryoswhich eventually developed into plantlets after transfer toauxin-free agar mineral medium containing sucrose. Cytokininssuch as benzyladenine (BA), zeatin, and kinetin induced compactgreen callus but in the absence of auxin failed to promote organogenesis.The interaction of IAA and kinetin resulted in the regenerationof the whole plant from stem explants. When NAA was used withkinetin, shoot development was totally inhibited and abundantroots were formed. Thus, the alternative morphogenetic eventsprobably reflect the biochemical subtleties occurring withinthe callus as a result of differences of actual endogenous levelsof growth substances in the tissues studied. These experimentshave been performed and interpreted on a histological basis.     

Plant regeneration from leaf-derived callus cultures of the tropical pasture legume Stylosanthes scabra Vog.

Callus cultures of 5 genotypes of S. scabra Vog. were optimally established from leaf tissue on Murashige and Skoog (MS) basal medium supplemented with 0.5–2.0 mg l^-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 1.0–2.0 mg l^-1 6-benzylaminopurine (BAP). On media containing 2, 4-D only, calli were soft, and rhizogenesis occurred on calli of 4 genotypes. Calli formed on media containing BAP only, but not with kinetin only. In the presence of 2, 4-D, BAP inhibited rhizogenesis and promoted better callus growth than kinetin. High frequency shoot induction was achieved for 3 genotypes on MS +2.0 mg l^-1 BAP. Roots formed on shoots when sub-cultured on half-strenght MS without growth regulators. The form of cytokinin used in the callus induction media appeared to affect subsequent shoot organogenesis. Genotypic differences were observed for shoot organogenesis. There was some morphological variation evident among regenerants.     

Cytological Studies on Tissue Culture of Pinus cembra

Callus tissue of Swiss stone pine, Pinus cembra L. var. sibirica Loud., has been grown successfully for 9 months through 7 transfers. Excellent initial growth was obtained from hypocotyl segments of 7-day-old seedlings. A complex agar medium was used supplemented with 2 mg/l 2,4-D, 0.05 mg/l kinetin, 1 g/l Edamin and 10% v/v coconut milk. No deterioration of growth was observed on this medium after numerous transfers. The callus tissue did not produce shoots or roots, but showed an ability for cytodifferentiation. Tracheids or tracheid-like structures were formed in every passage, but the tracheids of the primary callus culture differed markedly from those of the subcultures. The tracheids of the primary culture probably already existed in the hypocotyl, whereas those of the subcultures were formed in vitro. The callus tissue was mainly diploid during the period studied, and more than 86% of the mitoses were diploid. A few mitoses with tetraploidy or a higher ploidy also occurred, but the tissue did not show any tendency to polyploidization.     

Somatic embryogenesis and plant regeneration from the anther of Aconitum carmichaeli Debx

Anthers of Aconitum carmichaeli Debx. were used for callus induction. After the addition of 5 ppm 2,4-D and 1 ppm kinetin callus induction occurred over a period of 15 weeks. When calluses were subcultured on a medium containing 1 ppm 2,4-D for 12 weeks embryogenesis occurred. Mature somatic embryos developed normal shoots when transferred to basal medium inoculated with 1 ppm GA and 5 ppm BAP. Rooting occurred after the transfer of shoots to a new medium containing 0.5 ppm IAA and plantlets formed. The transplantation of these was successful and all plants matured during 5 months subsequent cultivation.     

In vitro Regeneration of Gerbera jamesonii Bolus from Leaf and Petiole Explants

Regeneration of adventitious shoots from leaf and petiole pieces of Gerbera jamesonii has been obtained on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins and cytokinins. About 75’77 per cent of the calli from both types of the explants produced 12’15 shoots per callus with 3 mg l^?1 SAP. Auxins and kinetin, separately failed to produce shoots. The shoots regenerated on the callus induction medium (elM). The regenerated shoots multiplied with 1 mg l^?1 SAP, were rooted on MS medium containing 1mg l^-1 BAP + 0.1 mg l^-1 IAA. The plants obtained were transferred to pots and acclimatized with 60’70 per cent success.     

Growth and Morphogenesis in Tissue Cultures of Anagallis arvensis

Morphogenesis has been induced in excised organs and callus tissue cultures obtained from various parts of the seedling and mature plants of pimpernel (Anagallis arvensis). Vigorously growing cell cultures capable of being periodically subcultured have been established in liquid as well as on the agar-solidified Murashige and Skoog's medium supplemented with 2,4-D (0.1 mg/1) + kinetin (0.1 mg/l) + coconut milk (10%). The callus tissue obtained from excised hypocotyl segments is white, soft, friable and fast growing, and has been subcultured over a period of two years without showing any sign of decline in growth. The optimum conditions for growth are at pH 5.9, temperature 27°C, and with 4% sucrose as the carbon source. Under appropriate nutritional supply these cultures can be manipulated to induce rhizogenesis in the suspension cultures, and buds and “embryo-like” structures on agar-solidified media. The excised leaves, hypocotyl and stem segments regenerate buds. Of the cytokinins used, 6-(y,y-dimethylallylamino)-purine proved to be the best for the number of cultures producing buds, as well as for the number of buds per culture. Anatomical studies revealed that buds arise from the epidermal and subepidermal layers of leaves and hypocotyl; these buds form shoots which eventually develop into plantlets.     

Preliminary observations on organogenesis inTaraxacum officinale tissue cultures

Summary Tissue cultures ofTaraxacum officinale have been isolated from the secondary thickened root. Callus development and leaf and root formation occur on a basal medium supplemented with coconut milk and IAA or NAA, and the addition of kinetin to these media enhances callus growth and organogenesis. Cultures grown on the basal medium with coconut milk and 2,4-D show only callus growth, but organogenesis is induced by the substitution of IAA for 2,4-D. In the 2,4-D grown callus a layer of secondary meristematic tissue is present and organogenesis apparently occurs from localized regions of this tissue which have undergone de-differentiation to the primary meristematic condition.     

Morphogenic studies in tissue cultures of the parasiteSantalum album L.

Whole seeds, excised embryos, and excised endosperm ofSantalum album were aseptically cultured with a view to studying seed germination in isolation from the host species, and to establishing callus cultures from both embryo and endosperm for comparative studies et their morphogenesis. Seed germination and seedling formation occurred normally only on modified White's medium supplemented with casein hydrolysate or coconut milk, or with both substances. Neither the excised embryo nor the endosperm grew on any of the culture media tested. However in about 17 per cent seed cultures on White's medium supplemented with 2,4-D, kinetin, and yeast extract, the endosperm degenerated, whereas the embryo callused and subsequently differentiated into innumerable embryoids; eventually the embryoids developed into normal plantlets. Callusing of the endosperm occurred also in seed cultures on four media supplemented variously with 2,4-D, kinetin, and yeast extract. Although the endosperm tissue grew through several passages no organ fornation was observed.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Habituation in Nicotiana bigelovii tissue cultures: Different behaviour of two varieties

The exceptionally high capacity for transformation to autotrophyfor hormones (habituation) discovered in Nicotiana bigeloviiled us to a comparison of the responses of 2 varieties (quadrivalvisand bigelovii) to different hormone treatments applied to theirseeds. Hormones used were: kinetin (0.8, 1.6, 3.2 ppm), 2,4-D(0.1, 0.4 ppm), IAA (2 ppm) and NAA (1, 2, 4 ppm). Callus formation and habituation were scored after transferon minimal medium (without hormones) 3 months after sowing.Var. quadrivalvis showed a much higher ability to form callusbut the callus formed was autonomous in a lower percentage ofcases when IAA or kinetin treatment was considered. Analysisof auxin-like substances in leaves of the 2 varieties showeda higher content in N. bigelovii var. quadrivalvis. Data arediscussed with particular regard to their relevance to tumorousphenomena in plants. ¹ Publication No 52 of the Laboratorio di Mutagenesi e Differenziamento,C. N. R. (Received March 30, 1971; )     

Somatic Embryogenesis and Plant Regeneration from Leaf Callus of Hordeum vulgare

Explants obtained from the basal portion of leaves of Hordeumvulgare (cv. Karan 92) gave rise to callus when cultured onMurashige and Skoog (MS) basal medium supplemented with 2, 4-dichlorophenoxyaceticacid (2, 4-D). Initially, the callus was friable, shiny-whiteand watery but subsequently some compact, nodular callus appeared.The latter were cultured on MS medium containing 0.05 mg l^–12, 4-D and 0.1 mg l^–1 N6-furfurylaminopurine (kinetin),when plantlets were generated. Histological studies showed thatplantlet regeneration occurred by the formation of somatic embryos.The regenerated plants had the normal diploid chromosome number(2n = 14). Hordeum vulgare, barley, somatic embryogenesis, tissue culture, plant regeneration