In vitro vegetative multiplication of Fuchsia hybrida

Rapid development of axillary buds from shoot-tips and nodes of 18 cultivars of Fuchsia hybrida was obtained on solid Murashige and Skoog medium with BAP (1 mg l^?1 and an auxin (0.1 mg l^?1). NAA as the auxin appeared to be more active than IAA or IBA. Vegetative shoots were subsequently isolated and developed up to 15 supplementary axillary shoots on the same solid medium. Agitated and non-agitated liquid media of the same composition were less effective. One-cm long shoots could be rooted in 20 days in the presence of IBA before being transferred to soil.     

In vitro propagation of garlic by shoot proliferation

Shoot buds (5–8 mm long), excised from dormant cloves of the New Zealand commercial garlic (Allium sativum L.) and a virus-free French cultivar ‘Rose-de-Kakylis’, proliferated both axillary and adventitious shoots on B-5 basal medium supplemented with 0.5 mg l^?1 isopentenyladenine (2-ip) and 0.1 mg l^?1 naphthaleneacetic acid (NAA). An 8-fold increase in shoot number occurred every 6 weeks. Shoots were readily rooted in B-5 + 0.01 mg l^?1 2-ip + 0.2 mg l^?1 NAA and transferred to pots, where about 70% of the shoots formed established plants. The plants raised by this shoot-proliferation method retained the diploid condition of the parents.     

In vitro propagation of ‘Troyer’ citrange from epicotyl segments

Isolated epicotyl, root meristem and root segment tissues of ‘Troyer’ citrange [Poncirus trifoliata (L.) Rat. × Citrus sinensis (L.) Osbeck] were established in continuous culture to compare their regeneration potential. Callus was obtained from these explants on a Murashige—Skoog (MS) medium containing NAA (10 mg l^?1) and BAP (0.1–10 mg l^?1). Formation of shoots from root segments was direct without callus formation on MS medium containing BAP (10 mg l^?1) and NAA (1 mg l^?1). Shoot formation from epicotyl callus occurred on MS medium containing 0.25 mg l^?1 BAP and 0.1 mg l^?1 NAA. Formation of shoots from epicotyl segments occurred on MS medium containing BAP (0.5 mg l^?1) and NAA (0.1–1.0 mg l^?1), while rooting of regenerated shoots occurred in treatments containing 2.0 mg l^?1 NAA alone. This system provides a rapid method for propagation of ‘Troyer’ citrange.     

Meristem culture and micropropagation of a variety of ginger (Zingiber officinale Rosc.) with a high yield of oleoresin

Summary

Meristems of ginger with or without leaf primordia were induced to form shoots on three-quarter strength Murashige-Skoog’s (MS) medium containing sucrose 6%, coconut milk (CM) 20%, ascorbic acid (AA) 100 mg l^?1, glutamine (GL) 400 mg l^?1, activated charcoal (AC) 250 mg l^?1, 6-benzylaminopurine (BAP) 0.5 mg l^?1, indolebutyric acid (IBA) 0.4 mg l^?1 and agar 0.8%. Meristem-derived shoots exhibited consistent multiplication on three-quarter strength MS medium containing sucrose (3%), AA (100 mg l^?1), AC (100 mg l^?1), BAP (4–5 mg l^?1) and agar (0.8%). Liquid media (agitated or static) were less effective than a solid (agar-gelled) medium for micropropagation. Kinetin and naphthalene acetic acid (NAA) incorporated at various levels (0.01–0.8 mg l^?1) with or without added BAP and IBA neither improved plantlet formation nor enhanced shoot multiplication. The in vitro plants were successfully established in vivo and the rhizome yield was comparable with that of plants grown by conventional methods.     

In vitro micropropagation from plumular apices of Turkish cowpea (Vigna unguiculata L.) cultivar Akkiz

Multiple shoot formation from the plumular apices excised from mature embryos of cowpea cv. Akkiz was obtained after pulse treatment with 10 mg/l BAP for 5 days followed by culture on MS medium containing 0.25, 0.50, 0.75 and 1.00, 1.25 mg/l BAP – with or without 0.10 mg/l NAA. Callus induction and shoot regeneration was recorded on all cultures containing BAP with or without NAA. However, inclusion of 0.1 mg/l NAA had positive effect on callus diameter and shoot length. Maximum mean number of 7.11 shoots per explant were obtained on MS medium containing 1.00 mg/l BAP. Longer shoots were recorded on MS medium containing various concentration of BAP+ 0.1 mg/l NAA compared to those containing various concentrations of BAP singly. All shoots cultured on MS medium containing 1 mg/l BAP were rooted on MS medium containing 0.50 mg/l IBA. Rooted plants were acclimatized at room temprature in soil contained in pots. All plants flowered and set seeds in the greenhouse after 3 months.     

Callus and axillary-bud culture of Vitis vinifera ‘Sylvaner Riesling’

Healthy growth of serially subcultured callus of the grape Vitis vinifera cultivar ‘Sylvaner’ was obtained by incubation at 30° C in continuous light in a defined culture medium containing 2% w/v sucrose, 1.0 mg l^?1 1-naphthaleneacetic acid (NAA) and 0.2 mg l^?1 kinetin (K). Organogenesis was not induced in this callus by alteration in the absolute or relative levels of NAA and K.Continued shoot initiation was obtained by culture of axillary buds in a medium containing 10^?5 M Benzyladenine (BA). Plantlets could be generated from these shoot buds by transfer to media containing 10^?7 M BA or lacking a cytokinin.     

Micropropagation of guava (Psidium guajava L.)

Summary

Guava (Psidium guajava L.) is difficult to propagate using conventional asexual techniques, with most growers using seedling planting stock. However, these seedlings are highly variable. We therefore developed an in vitro technique to clonally propagate guava. Various concentrations of BAP (6-benzylaminopurine) and TDZ (thidiazuron) were used to regenerate and micropropagate plants. Two explant sources were compared: greenhouse grown plants (GHRP) and in vitro-harvested axillary buds (IVDS). GHRP with BAP at 2 mg l^–1 gave 3.7 shoots per single node cutting with an average length of 0.7 cm. Shoots 3.0 cm long were obtained with 0.5 mg l^–1 BAP, however only 2.1 shoots per explant were produced. For IVDS, the largest number of shoots (3.9 per explant) was obtained with BAP at 0.25 mg l–1 , with an average shoot length of 1.6 cm. Generally, lower concentrations of BAP gave fewer but longer shoots. The highest number of roots and longest roots per shoot (5.4 and 2.0 cm, respectively) were obtained with 1 mg l^–1 indole-3-butyric acid (IBA). A protocol for producing clonal plants over eight weeks is described.     

Castanea sativa plantlets proliferated from axillary buds cultivated in vitro

Chestnut plants were proliferated in vitro from axillary buds of juvenile shoots. N6-Benzyl-aminopurine (BAP) at 0.1?0.5 mg l^?1 was optimal for shoot multiplication. The important role played by the macronutrient formula on shoot multiplication, and especially on the rooting-stage, is emphasized. The MS ($\text{1}\text{2}$ NO₃) macronutrients gave the best rooting percentage as well as the highest number of roots per rooted shoot. In these experiments, shoots remained in the 3 mg l^?1 indole-3-butyric acid (IBA) medium for 12 days, after which they were transferred to an auxin-free medium where roots developed fully. Optimum rooting was achieved by immersing the 1 cm basal end of shoots in concentrated IBA solutions (0.5?1 mg ml^?1) for periods ranging from 2 to 15 min.     

Increased haploid production in Saintpaulia ionantha by anther culture

Anthers of Saintpaulia ionantha containing late-uninucleate stage pollen produced callus from the anther interior after 3–4 weeks culture on Murashige and Skoog medium supplemented with 1 mg l^?1 naphthylene acetic acid (NAA) and 0.5 mg l^?1 1,6-benzylaminopurine (BAP). Shoot regeneration occurred rapidly and up to 200 shoots could be recovered from callus derived from a single anther within 10 weeks. Examination of roottip mitoses from transplanted, established plants demonstrated that, with few exceptions, the plants were haploid, thus indicating that the callus was pollen derived. Exposure of buds to low temperature prior to anther excision was inhibitory to callus production. Shoot regeneration was studied by scanning-electron microscopy.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

In vitro propagation of Gladiolus by precocious axillary shoot formation

Axillary buds excised from corms of 3 cultivars of hybrid Gladiolus were cultured on nutrient medium containing various levels of 6-benzylaminopurine (BAP). BAP prevented dormancy, promoted shoot growth and inhibited root development. These effects were accompanied by precocious outgrowth of axillary shoots which developed into detachable, independent in vitro plantlets which could be serially sub-cultured for further proliferation. The level of BAP required to promote a steady rate of axillary branching was inversely related to its natural propagation rate. Plantlets not sub-cultured on to fresh medium eventually became dormant with the formation of small corms which could be planted in compost.     

Large-scale in vitro propagation of giant reed (Arundo donax L.), a promising biomass species

Summary

Giant reed (Arundo donax L.), a promising energy crop, is vegetatively-propagated from fragments of stems and rhizomes. This may limit large-scale cultivation, since it is time-consuming and involves considerable cost and effort. Tissue culture is an alternative to conventional methods of vegetative propagation and may represent a useful tool for large-scale propagation of plants for biomass production programmes. This report describes a protocol for the large-scale in vitro propagation of giant reed by adventitious bud formation. Stem nodes with dormant buds proved to be the most effective to initiate in vitro cultures giving the highest percentage of differentiated shoots (77%) compared to the other plant fractions tested. A sterilisation procedure using 5 g l^–1 HgCl₂ enabled the production of sterile explants. Moreover, early results indicated that late Autumn excision dates not only gave a higher percentage of well-developed shoots ( > 80%), but also a lower level of bacterial contamination (15 – 20%). 6-Benzylaminopurine (BAP) at 3.0 mg l^–1 was most effective in promoting shoot multiplication when added to a basal medium containing Murashige and Skoog (MS) macro- and micro-nutrients, Morel’s vitamins, 30 g l^–1 sucrose, and 7.0 g l^–1 bacteriological agar supplemented with 1.0 mg l^–1 indole-3-acetic acid (IAA) and 0.05 mg l^–1 gibberellic acid (GA₃). Rooting was successfully induced on the same basal medium used for proliferation, modified by halving the MS macro-nutrients and replacing BAP and the other growth regulators with 2.0 mg l^–1 1-naphthaleneacetic acid (NAA). Successful acclimatisation (> 95% survival) of plantlets was carried out, even in late Winter, in a cold greenhouse or under simpler facilities such as shade nets.     

Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.     

Callus induction and plant regeneration from cotyledonary explants of ash gourd (Benincasa hispida L.)

A protocol for the production of complete plantlets through multiple shoots from the cotyledon-derived calli of ash gourd (Benincasa hispida L.) is described. The embryos were excised from mature seeds and cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurin (BAP, 1–5 μM). After 10 days the well-developed green cotyledons from the growing embryos were isolated and cultured on MS medium fortified with 2,4-D (1–6 μM). The cultured cotyledons gave rise to luxuriantly growing calli after 6 weeks. These calli were subcultured on MS medium supplemented with various concentrations of BAP (1–6 μM) alone or in combination with naphthalene acetic acid (NAA, 0.2 and 0.5 μM) for regeneration. The regenerated shoots were multiplied and rooted on quarter strength MS medium supplemented with indole-3-butyric acid or NAA (1–5 μM). The rooted shoots were transplanted to soil with 90% success.     

In vitro regeneration of Camellia reticulata by somatic embryogenesis

Camellia reticulata L. plantlets were regenerated by direct and indirect somatic embryogenesis from immature zygotic embryos. Initial explants (cotyledon sections and embryonic axes) produced somatic embryos without intermediate callus tissue when grown on Murashige and Skoog’s basal medium with 30 gl^-1 sucrose and no growth regulators; the somatic embryos completed their development in 4-6 weeks in the same medium. Embryogénie competence was increased by 0.5 and 1 mg l^-1 IBA. Histological observation showed the embryos to originate from epidermal and subepidermal cells of the cotyledon and hypocotyl explants. Secondary somatic embryos developed directly from the cotyledons and hypocotyl region of primary somatic embryos by a process that was morphologically very similar to that occurring on zygotic explants. Direct repetitive embryogenesis was maintained by this system. Up to 40% germination occurred when mature somatic embryos were isolated and incubated in medium supplemented with 1 mgl^-1 GA₃ + 1 mgl^-1 IAA. Indirect somatic embryogenesis was induced in callus differentiated on cotyledon explants after three months’ culture in media containing IBA or NAA and/or BAP, embryogenic capacity being retained by callus subcultured on 0.5 mg l^-1 IBA + 1 mg l^-1 BAP.     

Influence of high concentrations of cytokinins on the production of somatic embryos by germinating seeds of tomato,aubergine and pepper

Summary

Somatic embryos of tomato, aubergine and pepper were initiated from intact seedlings when seeds were cultured on medium containing 6-benzylaminopurine (BAP) or thidiazuron (TDZ). The percentage of explants producing somatic embryos was highest for aubergine on media containing low concentrations of BAP, i.e. 0–10 mg l^–1, for tomato at 15–20 mg l^–1 and for pepper at 40–80 mg l^–1. Aubergine and tomato produced fewer somatic embryos per responsive seedling when cultured on medium containing TDZ, and pepper did not produce any somatic embryos on media containing 0–20 mg l^–1 1 TDZ. Morphogenesis of the seedlings producing somatic embryos was similar for all the genotypes, i.e. the seedlings were dwarf, only the cotyledons expanded, development of the apical meristem and the root were suppressed and a ring-like crown of nodular tissue developed at the base of the hypocotyl from which somatic embryos were initiated. Co-cultivation of tomato and aubergine seeds with seeds of pepper in media containing 0, 5, 10, 15 and 20 mg l^–1 BAP inhibited somatic embryogenesis in tomato and aubergine instead of assisting somatic embryogenesis in pepper. This is discussed in relation to the recent findings for the induction of somatic embyrogenesis in peanut (Arachis hypogea L.) and the role of BAP and TDZ.     

Direct shoot organogenesis on hypocotyl explants from in vitro germinated seedlings of Psidium guajava L. cv. Allahabad Safeda

Multiple shoots were generated via direct organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45-day-old) of Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without α-naphthalene acetic acid (NAA) were tried. Thidiazuron (0.1 μM) along with 0.54 μM NAA gave the highest response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS supplemented with 2.22 μM BAP + 2.32 μM kinetin. The regenerated microshoots were elongated using a quick dip of gibberellic acid (GA₃; 1.44 mM) prior to culture on MMS medium supplemented with 0.88 μM BAP and adenine sulphate (54.29 μM) for 2 weeks. Rooting of microshoots was achieved best on half strength MMS medium supplemented with 4.90 μM indole-3-butyric acid along with 100 mg l⁻¹ activated charcoal.     

In vitro germination of walnut (Juglans regia L.) embryos

The present studies were undertaken with a view to standardize the medium and culture conditions for embryo culture of five cultivars of walnut viz., ACO 38853, Netar Akhrot, Gobind, Solding Selection and Blackmore. Embryos from mature fruits were aseptically excised and cultured on MS medium supplemented with different combinations of BAP, kinetin and GA₃. Best performing medium was MS with 0.5 mg l⁻¹ kinetin, 0.5 mg l⁻¹ BAP and 2 mg l⁻¹ GA₃ yielding 66.6% germination in Netar Akhrot after 12 days of culturing. Percent germination of excised embryos was higher when GA₃ and cold treatments were simultaneously applied as compared to those when applied separately. Netar Akhrot was found to be the best responding cultivar, which had a range of 25–66.6% embryo germination under different culture conditions. Plantlets with shoots and roots have been obtained in Netar Akhrot and ACO38853 and are transferred to soil after hardening.     

香雪兰种子胚的组织培养和植株再生

香雪兰的成熟胚和未成熟胚切段，在含有ＩＡＡ２ｍｇ／Ｌ、ＮＡＡ０３ｍｇ／Ｌ和ＢＡＰ０５ｍｇ／Ｌ的改良Ｎ６培养基上可形成半透明的瘤状愈伤组织。将这种愈伤组织培养于含有ＮＡＡ０３ｍｇ／Ｌ、ＢＡＰ０５ｍｇ／Ｌ和ＫＴ０５ｍｇ／Ｌ的ＭＮ６培养基可分化出丛生芽。生根培养基为含ＮＡＡ０５ｍｇ／Ｌ的改良Ｎ６培养基。相同的外植体可在ＭＮ６＋ＩＡＡ２０ｍｇ／Ｌ＋ＢＡＰ３０ｍｇ／Ｌ培养基上通过体细胞胚胎发生途径直接形成小植株。讨论了外源激素对香雪兰种子胚离体培养的作用。     

In vitro regeneration from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit

Methods to regenerate whole plants from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit were established. To optimize shoot induction, leaf explants were cultured on media containing different types and combinations of plant growth regulators. Growth was initiated within 17–24 days culture, and included callus formation, and root or shoot organogenesis ranging from 20 to 100% regeneration. Shoots were induced only when explants were cultivated on MS medium containing a combination of NAA and kinetin, NAA and BAP, IAA and Kinetin, or IAA and BAP. On media containing NAA and BAP, dark incubation was critical for efficient direct shoot regeneration from explants. Direct shoot formation and the highest number of shoots per explant (17.6) were obtained from leaf explants cultured in the dark for 30 days on MS medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ BAP. Shoots cultured on MS medium containing 0.1 mg l⁻¹ NAA formed tuberous roots with microtubers within 42 days. Healthy regenerated plants were acclimated and transferred to a greenhouse.