Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. & C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5–8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30–35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea—an endangered medicinal plant.     

Regeneration via somatic embryogenesis and organogenesis in different cultivars of cotton (Gossypium SPP.)

Summary In vitro studies related to somatic embryogenesis and organogenesis were performed in different cultivars of cotton. Gossypium hirsutum cultivars SH-131, LH-900, Hybrid H8, Khandwa-2, and Gossypium arboreum cultivars BD-1, BD-6, Sarvottam, and Jawahar Tapti were screened for their ability to regenerate in vitro. Cotyledonary leaves and hypocotyls were used as explants. Vigorous callusing was observed in G. arboreum cultivars. Globular somatic embryos were formed in BD-1, BD-6, Sarvottam, Jawahar Tapti, SH-131, and LH-900. Heart-shaped and torpedo stages were also observed. Embryos of BD-1 and BD-6 germinated and formed distincts shoot and root poles. 2-Isopentenyladenine (2iP) was effective in the induction of somatic embryos. Hybrid H8 and Khandwa-2 regenerated by directly forming shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine and formed roots on hormone-free MS medium     

亚洲百合花丝离体培养器官形成的细胞形态学研究

对亚洲百合的花丝进行离体培养,并利用常规石蜡制片技术对诱导效果最好的材料进行细胞形态学观察,研究花丝在离体培养过程中器官形成的细胞形态学变化。结果表明:花丝在MS+BA0.5 mg/L+NAA0.5 mg/L的培养基上诱导效果最好。离体培养后其形态学下端切口内方的1～3层细胞首先启动脱分化,然后是内方的10～12层细胞,而其他部位的细胞自始至终未启动脱分化。亚洲百合的再生方式为器官发生型,器官通过胚性愈伤组织间接产生,在胚性愈伤组织团表面附近形成芽原基,或在胚性愈伤组织团内部形成根原基,有时同时分别在内、外形成根原基和芽原基后再通过维管组织连接成完整的植株。本研究为亚洲百合的人工调控提供基础理论依据。     

Regeneration and plantlet development from somatic tissues of <Emphasis Type="Italic">Aristolochia fimbriata</Emphasis>

Aristolochia fimbriata is a small herbaceous perennial in the basal angiosperm family Aristolochiaceae. The family contains diverse floral forms ranging from radial to monosymmetric flowers with a wide variety of insect pollinators. Additionally, Aristolochia species contain secondary metabolites that are important natural toxins and traditional medicines, and are critical to the reproduction of swallowtail butterflies. These characteristics, in combination with the small genome size and short life cycle of A. fimbriata, have prompted further development of this species as a model system to study the evolution of basal angiosperms. As a prerequisite for developing a genetic transformation procedure for Aristolochia, we developed protocols for in vitro plant multiplication, shoot organogenesis, rooting, and acclimation of tissue culture-derived plants. Two varieties of Aristolochia were multiplied in vitro and rooted with 100% efficiency. Shoot regeneration was achieved within 1 month of culture initiation from whole leaf, internodal stem, and petiole explants. The highest regeneration success (97%) was recorded for stem explants. Regenerated and rooted shoots were acclimated to greenhouse conditions and developed flowers within 4 weeks of transplanting.     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

吉祥草的花部器官发生及其系统学意义

利用扫描电镜（SEM）观察了吉祥草（Reineckia carnea）（铃兰科）的花部器官发生发育过程。吉祥草花被片、雄蕊的发生方式是由近轴端向远轴端发生的逆单向型（reversed unidirection）,花发育后期花被片合生形成花被筒,花丝与之贴生。伴随花被片、雄蕊发生,三枚心皮也由近轴向远轴方向相继发生,随后彼此合生发育。花序顶部的花易发生花器官数目变异。结合早期花原基形态以及花器官数目变异情况分析,吉祥草的花被片与雄蕊可能是由共同原基分化而成。从花部器官发生式样和花被筒形成时间两方面比较吉祥草属、白穗花属和铃兰属的特征发现,三属中,铃兰属处于相对进化的位置,而白穗花属比吉祥草属更为原始。     

In vitro regeneration of adult Pinus sylvestris L. trees

The propagation of adult conifer trees by tissue culture has been studied for the last twenty years, but problems related to the juvenile to adult phase change of trees have limited the practical applications of these tissue culture procedures. This paper describes a micropropagation protocol for the in vitro propagation of mature Scots pine trees. In this study, dormant shoot buds, which had not started to elongate, were collected from twenty-one adult Pinus sylvestris trees (> 15 years old) during the winter. The sampled buds were cut transversely into slices of 0.5 to 1 cm in thickness and were cultured on three types of culture media (DCR, WP and LPm) supplemented with four cytokinins (BA, mT, Tdz and Z), at two different concentrations (25 and 50 µM), except for Tdz, whose concentrations were diluted to 5 µM and 2.5 µM. The evaluated culture media did not show significant differences in the bud organogenesis capacity. In fact, the highest organogenic response was obtained with buds cultured on DCR and WP media and by explants cultured on medium supplemented with 25 µM meta-topolin. This protocol is a successful and efficient biotechnological approach to the micropropagation of adult P. sylvestris trees.     

Pentamerous flowers in the genus Phytolacca have been derived from trimerous flowers—New evidence from the floral organogenesis of Phytolacca dodecandra

The floral organogenesis of Phytolacca dodecandra L′Her. (Phytolaccaceae) has been observed under both scanning electron microscope (SEM) and light microscope. The primordia of the floral appendage are arranged according to a pentamerous pattern and acropetal succession. Five sepal primordia arise in a 2/5 sequence, and no petal primordia have been observed. The stamen primordia arise centrifugally. The first two pairs arise successively opposite sepal one and two. In the subsequent initiation of inner and outer stamens, P. dodecandra differs from other species in the genus Phytolacca. The four or five carpel primordia arise in rapid succession, usually equal in number and alternating with the inner stamens. The effects of temporal and spatial factors during the floral organogenesis of P. dodecandra are discussed. The data on the androecial ontogeny in P. dodecandra refute the existence of diplostemony in Phytolaccaceae, in which P. dodecandra occupies a pivotal systematic position. The androecial ontogeny in P. dodecandra supports the viewpoint that in the genus Phytolacca pentamerous flowers have been derived from trimerous flowers.     

球茎甘蓝花托及花柄切口直接出芽的研究

培养球茎甘蓝（ Ｂｒａｓｓｉｃａ ｃａｕｌｏｒａｐａ）的带有花托、花柄和子房的外植体。在附加 Ｂ Ａ 和 Ｇ Ａ３ 的培养基上,花托部位直接出芽;在附加不同浓度 Ｂ Ａ 的培养基和附加 Ｂ Ａ 和 Ｇ Ａ３ 的培养基上均诱导花柄切口直接出芽,在附加 Ｂ Ａ 和 Ｎ Ａ Ａ 的培养基上,花托花柄切口剧烈增生愈伤组织。组织细胞学观察了芽的发生过程,结果表明：花托芽是由靠近维管束的多个皮层薄壁细胞恢复分裂能力形成,并与母体建立维管联系;花柄切口出芽是由皮层几个相靠近的薄壁细胞恢复分裂能力共同形成,与母体没有维管联系;花托增生愈伤组织也是皮层薄壁细胞剧烈分裂的结果。     

Hormonal and histological studies related to in vitro banana bud formation

Shoot apices of Musa subgroup AAA `Grande Naine' were used for in vitro culture establishment. The endogenous hormone levels and their effects on bud formation were evaluated during a 75-day period. Cytokinins, IAA and ABA were separated by HPLC and quantified by means of ELISA. Enzymatic degradation of IAA was determined by the colorimetric method. Explants were maintained on establishment medium for 60 days. The endogenous cytokinins were higher in the basal portion of the explant. Subculture to proliferation medium (65 to 75 days) resulted in a substantial increase of cytokinins in the basal portion and in a decline in the apical portion. 2iP was the predominant cytokinin in the tissue. The endogenous level of IAA and the IAA/cytokinin ratio decreased after the 65th day of culture. The level of ABA was reduced from the time of inoculation up to the 75th day of culture. Histological analysis indicated that buds formed at the leaf base at the 65th day of culture. This revised version was published online in August 2006 with corrections to the Cover Date.