Efficient In Vitro Shoot-regeneration Systems in Cassava (Manihot esculenta Crantz)

Two different protocols for in vitro regeneration of cassava using zygotic embryos and nodal axillary meristems have been developed. In both cases, buds were regenerated directly from excised explants without an intervening callus phase after a two-step culture procedure. In cotyledonary explants derived from zygotic embryos, prolific shoot formation occurred within 2—3 weeks on MS medium supplemented with 0.5—5 mg/1 BAP alone or in combination with 0.1 mg/1 NAA. Nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on MS medium containing 10 mg/1 BAP. These latter structures, when cultured on MS medium supplemented with 0.1 mg/1 NAA, 1 mg/1 BAP and 0.1 mg/1 GA₃, produced multiple shoots. Somatic embryos isolated at the globular/torpedo stage from zygotic embryo explants were also capable of multiple shoot production on medium with 1 mg/1 BAP. Rooting of regenerated shoots exceeded 95 % in phytohormone-free MS medium. No change in their ploidy levels was observed. Therefore, the protocols developed should be of use in the particle gun and Agrobacterium-mediated genetic transformation of cassava.     

Plant Regeneration from the Hybridization of Brassica juncea and B. napus Through Embryo Culture

Crosses were made to produce interspecific hybrids between Brassica napus × B. juncea and their reciprocals with the aid of embryo culture techniques. A better response of hybrid embryo culture was obtained from two cross combinations of B. juncea × B. napus (Ames 24521 × Huyou 15 and Vittasso × Zheshuang 72) than from their reciprocals. Embryo culture was more effective in terms of plant regeneration when embryos were cultured in vitro at 15 days after pollination (DAP), while more calli were initiated when embryos were excised and cultured at 10 DAP. A better response was observed on the MS medium with 0.3 mg l^?1 naphthylacetic acid (NAA) + 1.5 mg l^?1 6‐benzylaminopurine (BAP) and with 0.3 mg l^?1 NAA + 2.0 mg l^?1 BAP. Callus formation and plant regeneration on these two media reached 55.43 and 26.65 %, and 66.98 and 24.61 %, respectively.     

Somatic Embryogenesis in Hypocotyl Protoplast Culture of Rapeseed (Brassica napus L.)

By using a system of agirose plating and agarose bead culture, it was possible to induce efficient somatic embryogenesis in protoplast-derived calli of two rapeseed varieties, ‘Ceres’ and ‘Duplo’. Protoplasts were isolated from hypocotyls. For the initial protoplast culture a modified 8P medium was employed containing 2,4D (1.0 mg/l), NAA (0.1 mg/ 1), BAP (0.4 mg/l) and mannitol (7 %). After microcalli were obtained in four weeks, somatic embryos were induced by a two-step method. This involved a modified MS medium containing 2,4D (3.0 mg/l) in the first step and no 2,4D, but BAP (3.0 mg/l) and GA₃ (0.1 mg/l) in the second. This procedure also secured plant regeneration.     

Role of Nitrogen and Gibberellin (GA3) in the Regulation of Enzyme Activities and in Osmoprotectant Accumulation in Brassica juncea L. under Salt Stress

Salt stress alters a wide array of plant metabolic mechanisms. Different strategies of the application of nutrients and phytohormones are required to overcome the adverse effects of salt stress. The main objective of the present study was to determine if added nitrogen (N) and gibberellin (GA₃) in growth medium could alleviate the adverse effects of salt stress on plant metabolism. Two‐week‐old plants were fed with: (i) 0 mm NaCl + 0 mg N kg^?1 sand + 0 m GA₃ (control), (ii) 100 mm NaCl + 0 mg N kg^?1 sand + 0 m GA₃, (iii) 0 mm NaCl + 40 mg N kg^?1 sand + 0 m GA₃, (iv) 0 mm NaCl + 0 mg N kg^?1 sand + 10^?5 m GA₃, (v) 100 mm NaCl + 40 mg N kg^?1 sand + 0 m GA₃, (vi) 100 mm NaCl + 0 mg N kg^?1 sand + 10^?5 m GA₃, (vii) 100 mm NaCl + 40 mg N kg^?1 sand + 10^?5 m GA₃. Growth and physio‐biochemical attributes i.e. shoot length, leaf area, fresh weight, dry weight, net photosynthetic rate, stomatal conductance, malondialdehyde concentration, electrolyte leakage, total chlorophyll concentration, nitrate reductase and carbonic anhydrase activities, proline and glycinebetaine concentration, leaf – N, potassium (K) and sodium (Na) concentration and K/Na ratio were affected by NaCl treatment. But application of N or GA₃ alone as well as in combination proved beneficial in alleviating the adverse effects of salt stress on these growth and physio‐biochemical parameters. However, N applied with GA₃ proved more effective than N and GA₃ applied alone. The results revealed that combined application of N and GA₃ may ameliorate most of the attributes and prove to be a physiological remedy to increase the tolerance against the ill effects of salt stress in Brassica.     

Plant Regeneration from in vitro Cultured Anthers of Black Mustard (Brassica nigra Koch)

Anthers of Brassica nigra, excised from fresh as well as cold-pretreated (3 days at 3 ± 2°C) buds cultivated on modified B₅ medium (Gamborg et al. 1968) containing sucrose level varying from 2 % to 10 %, along with 1O^?6M BAP (benzylaminopurine) and 9 × 10^?6M 2,4-D (2,4-dichlorophenoxyacetic acid), developed calli and/or embryos. The latter response was observed only in anthers reared on media containing 6 % or higher levels of sucrose. On media containing two or four per cent sucrose, the anthers produced calli, exclusively. The growth of embryos was inhibited or else they started callusing if left on the media containing higher levels of sucrose. However, on transfer to MS medium (Murashige and Skoog 1962), containing 2 % sucrose, embryos started callusing and subsequently a few secondary embryos differentiated. Such embryos were sub-cultured on MS + 5 × 10^?6M BAP + 2 % sucrose, wherein numerous shoots developed from embryos. The shoots were rooted by transferring to a medium containing 5 × 10^?6M NAA (naphthalene acetic acid). Within two months of culture, some of these plants started flowering in vitro.     

Foliar application of the exogenous plant hormones at pre-blooming stage improves flowering and fruiting in cashew (<Emphasis Type="Italic">Anacardium occidentale</Emphasis> L.)

The problem of declining tree yield has led to an investigation into the effectiveness of foliar application of exogenous hormones to improve flowering, fruit set, and fruit retention in cashew. Five exogenous hormones, one Gibberellic Acid (GA₃) and four Auxins (IAA, IBA, NAA, and 2,4-D) at seven different rates of application (0 mg L⁻¹, 10 mg L⁻¹, 25 mg L⁻¹, 50 mg L⁻¹, 100 mg L⁻¹, 250 mg L⁻¹, and 500 mg L⁻¹) were tested on six yield-related components of the two Brazilian cashew genotypes. This trial was a factorial split-split-plot design with each treatment replicated five times within a tree and three replications (three trees) per genotype. Responses varied significantly between exogenous hormones, concentrations and genotypes. The cashew plants used showed hormone-specific and optimum concentration response patterns. Of the five exogenous hormones tested, GA₃ was most effective as its application at 50–100 mg L⁻¹ gave five-fold improvements in flowering (precocity and number of hermaphrodite flowers) and fruiting, and about 69% increase in fruit retention ability and 25% in nut size. Panicles treated with GA₃ also produced relatively bigger nuts compared to the untreated. Days to flowering was found to be hormone sensitive, while production of hermaphrodite flowers, fruit set, and nut development tended to be concentration specific. The GA₃ exhibited a broad concentration tolerance among the five exogenous hormones investigated. Our data showed that using GA₃ at 50 mg L⁻¹ will enhance flowering precocity, shorten flowering duration, increase production of hermaphrodite flowers and fruit set significantly, and resultant nuts develop optimally with high percentage retention. Thus, it suggests cashew yield could be increased by exogenous foliar application of GA₃ at 50–100 mg L⁻¹ at pre-blooming stage.     

Production of two mandarin × trifoliate orange hybrid populations via embryo rescue with verification by SSR analysis

Intergeneric sexual hybridizations were conducted between two genera of Rutaceae, with Satsuma mandarin (Citrus unshiu Marc) and Red tangerine (C. reticulata Blanco) as maternal parents, and Poncirus trifoliata (L.) Raf as the paternal parent, in an effort to generate hybrid populations for both molecular mapping and rootstock breeding. Embryo rescue is important for citrus sexual breeding because polyembryony can interfere with hybrid embryo recovery. Immature embryos of 80, 85, 90 days after pollination (DAP) from the Satsuma mandarin (S) × trifoliate orange (P) cross, 80 and 85 DAP from the Red tangerine (R) × trifoliate orange (P) cross, were cultured on MG1.0 medium consisting of MT basal medium supplemented with 1.0 mg l⁻¹ GA₃ and 4% sucrose. The results showed that 80 DAP was the optimal time for embryo rescue of the tested crosses, as evidenced by embryos at this stage exhibiting high germination rates, 37.3% for S × P and 51.3% for R × P. Among the eight tested media, MT medium supplemented with 0.5 mg l⁻¹ GA₃ was the best one for Satsuma mandarin, and MT plus 1.0 mg l⁻¹ GA₃ for Red tangerine. A total of 85 plants were obtained from S × P, and 340 from R × P. Out of them, 44 progenies from S × P and 111 from R × P were sufficiently confirmed to be hybrids by morphological characterization and SSR analysis. In addition, two hybrid callus lines were obtained from S × P and R × P respectively.     

Genetic factors influencing the regeneration ability of rye (Secale cereale L.). II. Immature embryos

Summary Immature embryos of seven rye inbred lines were cultured on modified MS medium containing 3 mg/dm^–3 2,4-D. According to thein vitro response lines were divided into four groups: (1) those producing non-embryogenic callus (NEC) from above 30% of the embryos and having a high rate of non-responding (NR) embryos, (2) those producing non-embryogenic callus (NEC) from the majority of embryos, (3) those producing NEC by the majority of embryos with a high percentage of calli regenerating roots, (4) those producing embryogenic callus (EC) and regenerating plants by above 50% of the embryos. The inheritance of these response types was analysed in F₁, F₂, and F₃ generations of crosses of some lines. The results obtained indicate that EC production and both plant and root regeneration are determined by recessive genes whereas the reduced ability for NEC production most probably by dominant genes. The lack of response is controlled by at least two interacting genes.     

Influence of Warm Intervals on the Effects of Vernalization and the Composition of Phospholipid Fatty Acids in Seedlings of Winter Wheat

Excised embryos of winter wheat were submitted to a different number of vernalization (2 °C) and devernalization (20 °C) cycles of different lengths. In all treatments sum of cold and warm intervals was 50 and 10 days, respectively. The influence of different temperature conditions of seedling growth on the effectiveness of vernalization and correlations between effectiveness of vernalization and final content of phospholipid fatty acids were analyzed.
All indexes of generative development of plants (length of vegetative phase, % of generative plants, number of side shoots) showed the most effective was uninterrupted vernalization beginning immediately after excision of embryos. Increasing the number of 2 ° C/20 °C cycles rapidly depressed effectiveness of vernalization, which reached the minimum with the 8.3 days at 2 °C and 2 days at 20 °C cycle and then improved again as cycles became shorter and more numerous.
Correlations between indexes of generative plant growth and indexes describing composition of fatty acids in seedlings showed that from 50 % to 60 % of variability in the composition of phospholipid can be associated with changes in the degree of generative induction. A higher degree of generative induction was associated with a higher share of < 16, 16: 0 and 18:2 acids and a lower share of 18: 3 acid and also with a lower value of the 16: 0/< 16 ratio of phospholipids.
The relation between the generative induction of plants and the composition of phospholipid fatty acids may however be blurred when induction proceeds under conditions of alternate short spells of vernalization and devernalization temperatures.     

Systemic View on Heading and Overwintering: Are They Always Opposed?

The study investigated the cultivars of non‐obligatorily requiring vernalization plant Festulolium braunii and assessed the influence of non‐hardy reproductive and hardy vegetative structures on overwintering effect. The study was conducted taking into account systemic relations between these types of structures. The results show the cultivars differ according to the percentage of headed and overwintered plants, when the cultivars with the most abundant heading – ‘Felopa’ and ‘Sulino’ – are also better at overwintering. The positive correlation between heading and overwintering characteristics was also observed, what seemed to be a rather new finding. It can be explained by systemic effect: non‐hardy later reproductive structures induce the post‐generative regrowth of vegetative shoots, which during shorter days halt development and become potentially hardy. More detailed interpretation is also provided including discussion of causal mechanisms of the detected phenomenon. The authors suppose that these mechanisms constitute a survival strategy for such perennial plants. The observed late heading which represents reproductive structures could be applied in plant breeding as a marker of winter‐hardiness among perennial grass plants which non‐obligatorily demand vernalization.     

Enhanced pollen grain embryogenesis and plant regeneration in anther cultures of Brassica juncea cv. PR-45

Summary Pollen grain embryogenesis in anther cultures of Brassica juncea cv. PR-45 was considerably enhanced by treating the donor plants with 4 mll^-1 (v/v) of ethrel or delayed sowing of the donor plants, the latter treatment being superior. The anthers derived from plants sown about two months after the normal sowing period showed 18% androgenesis as compared to 3.5% in the control.Pollen grain embryos normally showed very poor germination (10%) on B₅ or B₅ containing GA₃. However, ABA or cold treatment promoted normal germination of these embryos. Exposure of the embryos to 4°C for 6 days, which proved to be the best treatment, induced 66% germination of the embryos.     

Effect of embryo age and culture media on plant growth and vernalization response in winter wheat

Summary Embryo age and composition of nutrient medium affected plant growth and response to vernalization in winter wheat (Triticum aestivum L.). Root and shoot development was more in older than in younger excised embryos, and more in a medium without kinetin than in one with kinetin. Kinetin (2 mg/l) in the medium did not accelerate vernalization, probably because it tended to inhibit seedling and plant growth.Embryo age and media did not completely replace vernalization. Twenty- and 16-day-old embryos responded by flowering after 4 weeks of vernalization. Among plants raised on a standard medium from 20-day-old embryos and vernalized for 4 weeks, 84.2% flowered by or before 50 days after transplanting. Time from embryo culture to heading for 20-day-old embryos with-4-week vernalization averaged 84.6 days. Immature embryos (16–20 days old) needed only 4 weeks of vernalization compared to 6 weeks for mature embryos. Excised embryos could be vernalized as efficiently as seedlings raised by embryo culture. Embryo culture at 16–20 days after anthesis coupled with 4-week cold treatment shortens generation time of winter wheat by about 40 days.Contribution No. 82-131-j, Department of Plant Pathology, Kansas Agricultural Experiment Station, Manhattan, KS 66506, USA.     

Interspecific hybridization between Cicer arietinum and C. pinnatifidum

An interspecific hybrid between Cicer arietinum cv. GL 769 and a wild species C. pinnatifidum was obtained after emasculation, pollination and application of growth regulators. Ovules were cultured and embryos were later dissected to obtain hybrid plants. These plants were albinos and morphologically resembled C. pinnatifidum. Shrivelled seeds were also obtained in 2% of the crosses, which on germination gave rise to albino plants. These plants did not survive beyond 20 days. The hybrid nature of these plants was confirmed by esterase isozyme studies. Hybrid shoots obtained from germinating embryos were cultured on modified ML-6 medium with BAP 2 mg/1, IAA 0.5 mg/1, where they turned green after 3–4 weeks. Transmission electron microscopy (TEM) studies on leaf sections from green hybrid shoots showed an improvement in the chloroplast structure, with better organized grana.     

Alleviation of drought stress in maize by exogenous application of gibberellic acid and cytokinin

The study was conducted to investigate the ameliorative roles of GA₃ and CK on adverse effects of drought in maize. Drought stressed maize plants were applied with GA₃ and CK at 50, 100, and 150 mg L^?l as foliar spray at the vegetative and the reproductive stages. Plant height, internode length, stem diameter, leaf chlorophyll index, and dry matter production were significantly affected by drought. In most cases, GA₃ and CK significantly improved the depressed plant traits, but in varying degrees depending on the growth stage encountering hormones, and their types and concentrations. Both GA₃ and CK were found to be very effective in alleviating drought-imposed adverse effects on maize at the vegetative phase. Such alleviating effects varied depending on the concentration of the hormones. Application of CK at 150 mg L^?l was excellent resulting in a 106% yield advantage compared to drought stress and 79.9% increase relative to well-watered controls. Conversely, GA₃ at 50 mg L^?l performed well showing 78.8% increase in grain yield. However, both GA₃ and CK had very little effect on improving the depressed growth and yield attributes in maize at the reproductive phase. The relative yield advantages for the hormones were mainly attributed to improving the cob and seed-bearing capacity of drought-stressed maize plants.     

Induction of Generative Development of Winter Rape (Brassica napus L. var. oleifera) in Relation to Vernalization Conditions and Age of Vernalized Plants

Germinating seeds and young winter rape plants were vernalized 56–63 days at 5 or 2°C under nine-hour days or in darkness. The highest percentage of generative plants and the most rapid flowering were obtained following the vernalization of young seedlings and germinating seed under conditions of nine-hour day, at 5°C. The least effective induction of generative development followed the vernalization at 2°C in continuous darkness. The vegetation period from the end of vernalization till the beginning of flowering was the shortest when four-week-old plants were vernalized under conditions of nine-hour day, yet the vegetation period from the beginning of germination to flowering was the shortest when seed germinating under conditions of nine-hour day were vernalized. The period was extending as older plants were being vernalized. Data indicating that the optimal temperature for vernalization of older plants is higher than for germinating seed and young seedlings were obtained.     

Effect of Prohexadione Calcium on Growth Components and Endogenous Gibberellins Contents of Rice (Oryza sativa L.)

Prohexadione calcium (Pro‐Ca) is a novel plant growth regulator that inhibits the late stages of gibberellins (GAs) biosynthesis in plants, thus reducing the vegetative growth of the plant. Pro‐Ca was applied to field‐grown rice cultivar Ilpumbyeo to evaluate its effect on growth and yield components and endogenous GAs, when applied at different rates and timings. This study illustrated that Pro‐Ca application decreased the internode length, stem length, panicle length, shoots fresh weight and stem lodging index, while stem breaking force was increased in all treatments. The effect of Pro‐Ca was more pronounced in 20 ppm concentration than 10 ppm. The number of panicles m⁻², ripened grain ratio and 1000 grain weight and milled rice yield parameters, increased with the application of Pro‐Ca, though the number of grains per panicle decreased insignificantly. The endogenous bioactive GA₁ contents significantly decreased with the application of Pro‐Ca, as the GA biosynthesis was interrupted at later stages, when GA₂₀ conversion to GA₁ was blocked, thus drastically reducing GA₁ and GA₈ levels, but only insignificant changes were observed for intermediates from GA₁₂ to GA₂₀. The levels of the endogenous GAs in rice shoots were measured by GC‐MS‐SIM using ²H₂‐labelled GAs as internal standards. Present results also illustrated that early application of Pro‐Ca was more effective than its late application for effective control of different growth parameters and thus reducing lodging in rice plants.     

Quantification of endogenous gibberellins in two flax (Linum usitatissimum L.) cultivars during seed development

Eight gibberellins (GAs) were identified and quantified in extracts of seed of the flax, Linum usitatissimum L. cv. Hwanamama, Hwabaekama by GC-MS-SIM and Kovats retention indices. Four of these gibberellins are members of the early-C13-hydroxylation pathway (GA₅₃, GA₁₉, GA₂₀, and GA₁), and four are members of the non-C13-hydroxylation pathway (GA₁₂, GA₂₄, GA₉, and GA₄). Of these eight gibberellins, GA₂₄ was more abundant in yellow-seeded flax (cv. Hwanamama) than in brown-seeded flax (cv. Hwabaekama), whereas GA₉ was 8.3-fold more abundant in brown-seeded flax (cv. Hwabaekama) than in yellow-seeded flax (cv. Hwanamama). In both flax cultivars, non-C13-hydroxylation pathway was the main route leading to endogenous accumulation of GA₄ in the seed.     

Cytokinin and gibberellin delay regreening of spathe tissue of Zantedeschia ‘Best Gold’

Spathe regreening is a primary determinant limiting the postharvest quality for most hybrids of Zantedeschia, e.g. ‘Best Gold’, wherein the spathe commences regreening approximately two days after it is fully open. To identify an effective method to postpone the regreening, several synthetic plant hormones were evaluated for their influence on changes in color during regreening of discs excised from the spathe of ‘Best Gold’. The tested synthetic plant hormones included 6-benzylaminopurine (BAP), zeatin, N₁-(2-chloro-4-pyridyl)-N₃-phenylurea (CPPU) and GA₃ at concentrations up to 0.1 mM. A concentration of BAP at 0.5 mM resulted in phytotoxicity symptoms on discs. Subsequently, the effect of a combination of BAP and GA₃ on the regreening was examined. Application of the cytokinins (in particular BAP at 0.1 mM) or GA₃ alone resulted in a one to five day delay in regreening on the spathe tissue. The most effective treatment in delaying regreening was from the simultaneous application of GA₃ and BAP in a ratio of 1:1 (v:v) at 0.1 mM, wherein regreening was delayed for more than ten days. This treatment was then tested on the entire inflorescence, resulting in a delay in regreening for between seven and eight days.     

Effects of barley chromosome on heading characters in wheat-barley chromosome addition lines

Heading time in cereals is a composite character determined by vernalization requirement, photoperiodic sensitivity and narrow-sense earliness. To study the effects of added barley chromosomes on the heading characters in wheat, two sets of wheat-barley chromosome addition lines, i.e., ‘Betzes’ barley chromosomes 2H to 7H added to ’Chinese Spring‘ wheat (CS-Be2H to CS-Be7H) and ‘New Golden’ barley chromosomes 5H and 6H added to ‘Shinchunaga’ wheat (Shi-NG5H, Shi-NG6H), were examined for their heading characters. All barley chromosomes except Be6H affected vernalization requirement and/or narrow-sense earliness in CS or Shi. Be5H chromosome also slightly increased the photoperiodic sensitivity of CS. Shi-NG5H addition line showed significantly decreased vernalization requirement in comparison with Shi, whereas CS-Be5H did not show any difference from CS. The F₁ hybrid of the cross, Shi-NG5H × CS-Be5H, exhibited the same level of vernalization insensitivity as the Shi-NG5H addition line, and plants with and without a vernalization requirement segregated in a 1 : 3 ratio in the F₂ generation. These observations, together with previous reports, suggest that the decreased vernalization requirement in the Shi-NG5H addition line was caused by the presence of a major dominant gene for spring habit, Sh₂, located on the NG5H barley chromosome. Furthermore, this study revealed that the Sh₂ gene in barley has a similar but weaker effect than the wheat vernalization insensitive gene, Vrn1, on the vernalization response in wheat. This revised version was published online in July 2006 with corrections to the Cover Date.     

Actions for ex situ conservation of Gloriosa superba L. - an endangered ornamental cum medicinal plant

Factors affecting in vitro propagation and microtuberization were evaluated for Gloriosa superba L., an endangered ornamental cum medicinal plant having limited reproductive capacity. Surface sterilization of tuber explants with 0.1% mercuric chloride (HgCl₂) for 5 min eliminated the contamination effectively with highest survival rate. Among the various combinations used, Murashige and Skoog (MS) medium with 2.0 mg L^?1 6-benzylaminopurine (BAP) + 0.5 mg L^?1 α-naphthalene acetic acid (NAA) containing 3% sucrose with 16-h photoperiod exhibited the greatest in vitro tuberization (3.2) with the highest shoot regeneration frequency (90%). The longest tuber regeneration occurred on MS media containing 4% sucrose. Transfer of in vitro-regenerated shoots to half-strength MS medium with 1.0 mg L^?1 indole-3-butyric acid (IBA) + 0.5 mg L^?1 NAA showed maximum root induction (66.6%). The in vitro-grown plantlets were successfully acclimatized and transplanted to sterilized soil and sand mixture (3:1) in the glasshouse with 70% survival. The colchicine content was determined in the tubers of ex vitro plants by HPLC using the same retention time (1.5 min) as that of the standard colchicine. This revealed that the micropropagation protocol developed by us for rapid mass production could be used as raw material for colchicine extraction and provides a basis for germplasm conservation and genetic improvement of G. superba.