Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

Efficient in vitro direct shoot organogenesis and regeneration of fertile plants from embryo explants of Bambara groundnut (Vigna subterranea L. Verdc.)

An efficient protocol has been developed for direct shoot organogenesis from embryo axes derived from mature seeds of two different landraces of Bambara groundnut. Multiple shoots were initiated on several media containing different concentrations and combinations of benzylaminopurine (BAP) or thidiazuron (TDZ). Efficient regeneration occurred when the embryo axes were first plated for 6 days on a medium containing high concentrations of BAP (1 mg/l) and alpha-naphthaleneacetic acid (NAA, 1 mg/l) and then cut transversely and transferred onto a medium containing 1.5 mg/l BAP. Shoot regeneration frequency was 100% and from five to eight shoots per explant were obtained. The importance of using embryo explants and cytokinins in the culture media, with respect to controlling the development of a highly organogenic system, was demonstrated. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants without an intermediate callus phase. The regenerated shoots were rooted on a medium containing 1 mg/l NAA and then transferred to the greenhouse. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. All were morphologically normal and fertile. The short duration, high efficiency and low frequency of somaclonal variation of this system make it well suited for wider biotechnological applications of Bambara groundnut-a neglected and under-utilized crop.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

Effect of auxins and cytokinins on plant regeneration from hypocotyls and cotyledons in niger (Guizotia abyssinica Cass.)

Tissue cultures were established from hypocotyl and cotyledonary leaf segments ofGuizotia abyssinica Cass. on MS medium supplemented with various concentrations of auxins (IAA, NAA, IBA or 2,4-D) and cytokinins (KN or BA). Expiants cultured on media with cytokinins or in combination with auxins produced shoot buds. Maximum number of shoot buds (20–25 per culture) were differentiated from cotyledonary leaf segments on medium with 2 mg 1^-1 each of KN and IBA. Rooting of regenerated shoot buds was acheived on medium with NAA. The obtained plantlets were successfully transferred to soil.     

In vitro morphogenetic responses and plant regeneration from pepper (Capsicum annuum L. cv. Early California Wonder) seedling explants

Explants from 13-d old pepper (Capsicum annuum, L. cv. Early California Wonder) seedlings were cultured in Murashige and Skoog (MS) medium supplemented with different levels of 1-naphthalene acetic acid (NAA) and 6-benzylamino purine (BAP). Multiple shoot-buds proliferated from the cut surfaces of cotyledon, shoot-tip and hypocotyl explants in one month. The best NAA to BAP combinations (mg/l: mg/l) for multiple shoot-bud regeneration of the above three explant types were 0.1 ∶ 5.0, 0.0 ∶ 5.0, and 0.1 ∶ 10.0, respectively. Root explants did not express any new morphogenetic response in all hormonal combinations tested. Regenerated shoot-buds were excised from the explants and cultured in 1/2X or 1X MS medium supplemented with different levels of Indole-3-acetic acid (IAA) or NAA. When cultured in full strength MS medium with 0.5 mg/l IAA or 0.4 mg/l NAA, 70% of the buds rooted in one month. Plantlets were established successfully ex vitro under greenhouse mist and grown to maturity.     

Rapid clonal multiplication of Angelonia salicariefolia through tissue culture

Nodal explants of Angelonia salicariefolia were cultured on MS basal medium and induced to form shoots when supplemented with either Kn (1.0 mg/l) or BAP (1.0 mg/l). Rooted shoots were formed in response to Kn+NAA (1.0 mg/l+0.5 mg/l). Subcultures of the shoots of these cultures grown on the same medium supplemented with 0.5 mg/l of NAA, IAA or IBA, together with lowered concentrations of inorganic salts, induced root formation in 20–30 days. Up to 18×10³ plants were produced from one plant in less than a month. Successful transfer of regenerants into soil has been accomplished.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

In vitro propagation of ginger (Zingiber officinale Rosco)

An efficient and promising protocol for in vitro propagation of Zingiber officinale Rosco using sprouting buds was established. Sprouting buds were sterilized and cultured onto MS medium supplemented with different growth regulators. Augmentation of MS-medium with 4.5 mg/l BAP recorded the highest percentage of shootlets multiplication. Shootlets were highly rooted on half strength of B₅ medium supplemented with 1.0 mg/l NAA. The maximum percentage of acclimatization, hardening and rhizomes production of in vitro derived plants in greenhouse was 80–100%.     

Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana)

Opening of apical and axillary buds of mature Douglas-fir and sugar pine trees was obtained on a newly formulated basal medium (DCR) without growth regulators. Elongation of buds was observed on 1/2 strength DCR with 0.3% activated charcoal (DCR-1). In sugar pine, multiple shoots were obtained when explants on DCR with 0.5 mg/1 BAP for 5–6 weeks were transferred to DCR-1 medium. On subculture, axillary buds again developed when shoots were cultured on DCR with 0.2 mg/1 BAP for Douglas-fir and 0.5 mg/1 BAP for sugar pine. These buds were again elongated on DCR-1 medium. By subculturing 7–10 shoots of Douglas-fir and 2–3 shoots of sugar pine, over 100 shoots can be obtained in a year.Abbreviations BAP N⁶-benzylaminopurine - KN kinetin - NAA -naphthalene acetic acid - IAA Indole-3-acetic acid - MS Murashige-Skoog medium - WPM woody-plant medium     

麝香石竹花芽离体发育的阶段控制

在离体条件下,利用不同的培养基对麝香石竹顶芽外植体的花芽发育进行了阶段控制的研究.结果表明,麝香石竹的顶芽外植体在MS+KT1.0mg/L+IAA1.0mg/L+蔗糖3.0%+琼脂0.8%的Ⅰ级培养基上能被诱导花芽发育的启动;然后,将已诱导花芽发育启动的顶芽外植体,转接到MS+KT1.0mg/L+IAA0.5mg/L+蔗糖1.5%+葡萄糖1.5%+琼脂0.8%的Ⅱ级培养基上能进行花芽的进一步发育形成花蕾,且能从一个花蕾继续分化发育重新产生2—3个花蕾;把花蕾再转接到改良的MS+BA2.0mg/L+NAA0.2mg/L+蔗糖1.5%+葡萄糖1.5%+琼脂0.8%的Ⅲ级培养基上,培养一周后花蕾的花瓣张开,花朵全部开放.不同麝香石竹品种,诱导花芽发育启动的效果不同,Scania品种诱导效果最好.花芽发育初期可溶性蛋白含量较高,但随着花芽发育的进程而迅速下降,不同花芽发育时期的过氧化物酶活性均强于营养器官.本文为花芽分化发育机理的研究创造了条件,也为鲜花生产探索了新路子.     

The Influence of Different Cytokinins and Auxins on Flower Neoformation in Thin Cell Layers of Nicotiana tabacum L.

Neoformation of flower buds was studied in thin cell layersderived from floral branches of Nicotiana tabacum L. Organogenesiswas found to be expressed in different ways according to thekind of auxin and cytokinin present in the culture medium. Inthe presence of synthetic auxins (NAA, CTA), more flower budswere generated than with the natural auxins (IAA, IBA), if theculture medium was also supplemented with a cytokinin. Theseconditions also stimulated the development of the buds to flowersor inflorescences. The most active cytokinin was BA. Naturalcytokinins (zeatin, IPA) appeared much less active but led toremarkable development of roots together with flower buds. Anorganogenetic effect of 2,4-D was observed in this experimentalsystem, leading to the conclusion that organogenetic activityof cytokinins and auxins in thin cell layer cultures stronglydepends on the molecular structure of the growth regulators. (Received September 30, 1987; Accepted March 23, 1988)     

Somatic embryogenesis and plant regeneration from cell suspension cultures of Rajeli (AAB), an endangered banana cultivar

Rajeli (AAB), a commercially valuable Indian banana cultivar, is presently under the threat of extinction due to various diseases, warranting development of the strategies for its conservation and incorporation of disease resistance. This elite genotype was successfully regenerated in vitro via establishment of cell suspensions followed by somatic embryogenesis. The immature male flower buds were cultured on gelled Murashige and Skoog medium supplemented with 4 mg/l 2,4-D, 1 mg/l each of IAA and NAA, D-Biotin, 100 mg/l Malt Extract, 100 mg/l L-Glutamine and 30 g/l sucrose. Embryogenic calli with translucent somatic embryos were observed after 4 months of male flower bud culturing. Fine embryogenic cell suspensions were established in the liquid medium of same composition but with 45 g/l sucrose. Of the various sugars tested, maltose in the maintenance medium yielded better growth of plated cells as compared to sucrose, fructose and dextrose. Embryos differentiated at a high frequency and mature somatic embryos developed into plantlets on MS medium supplemented with 0.5 mg/l BAP. Approximately 40 % of the torpedo stage somatic embryos germinated and developed into complete plants. The regenerated plants exhibited normal phenotype during acclimatization in the greenhouse. The technique can be employed for conservation of this endangered cultivar and its improvement via mutagenesis and genetic transformation.     

Efficient in vitro regeneration of fireweed,a medicinal plant

Epilobium angustifolium L. (fireweed) is a medicinal plant that has been used to treat diarrhea, mucous colitis, irritable-bowel syndrome, skin problems, prostate problems, menstrual disorders, asthma, whooping cough, and hiccups. A highly efficient and rapid regeneration system via multiple shoot formation was developed for fireweed. Explants (leaf, petiole, root, and stem segments) excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. Explant browning, a major problem for regeneration, was overcome by adding 100 mg/l ascorbic acid to all prepared media containing growth regulator combinations. Root explants formed more shoots than other explants. Best shoot proliferation was obtained from root explants cultured on media with 0.1 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were transferred to rooting media containing different concentrations of IAA, IBA, NAA or 2,4-D. Most shoots developed roots on medium with 0.5 mg/l IAA. Rooted explants were transferred to vermiculate in Magenta containers for acclimatization and after 3 weeks they were planted in to plastic pots containing potting soil and maintained in the plant growth room.     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin     

In vitro morphogenesis from cotyledon and epicotyl explants and flow cytometry distinction between landraces of Bambara groundnut [<Emphasis Type="Italic">Vigna subterranea</Emphasis> (L.) Verdc], an under-utilised grain legume

The morphogenetic competence of Bambara groundnut was assessed for different landraces, explant sources and media compositions. With cotyledon explants, the best callusing occurred on a medium containing 3 mg/l BAP + 0.5 mg/l NAA, while roots were produced with 3–5 mg/l BAP + 0.5 mg/l NAA. Shoots regenerated (∼6%) from cotyledons on media with BAP alone (3–5 mg/l) or combined with 0.01–0.1 mg/l NAA. Flowers were regenerated on 5 mg/l BAP + 0.5 mg/l NAA, without any intervening callus phase. With epicotyls, the highest callusing was on 3 mg/l BAP + 0.5 mg/l NAA, and shoots regenerated (15–20%) on 3 mg/l BAP alone or with NAA at concentrations that depended on the landrace studied. Regenerated shoots rooted on hormone-free medium, and plants transferred to the greenhouse were all morphologically normal and fertile. Flow cytometry showed that most regenerants were diploid and in addition permitted to distinguish between landraces according to their relative nuclear DNA content. This is the first report on de novo regeneration in vitro of Bambara groundnut, an important yet neglected legume crop.     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside