Establishment of a high-frequency regeneration system in <Emphasis Type="Italic">Cerasus humilis</Emphasis>, an important economic shrub

Cerasus humilis is a species of small, perennial, drought-resistant and multipurpose deciduous shrub grown in arid and semi-arid conditions in northern China. In this study, an efficient protocol for the rapid micropropagation of C. humilis has been standardized using stem and/or leaf explants. Direct multiple shoot induction was observed when the stem explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The highest shoot induction was obtained when stem explants from adult trees were cultured on MS medium supplemented with 2.0 mg L^?1 6-benzyladenine (6-BA) and 0.9 mg L^?1 α-naphthaleneacetic acid (NAA). The leaf and stem explants cultured on MS medium with 1.0 mg L^?1 6-BA and 0.6 mg L^?1 NAA, and 0.5 mg L^?1 6-BA and 0.8 mg L^?1 NAA, respectively, produced the highest induction frequency of callus. Maximum proliferation of callus was observed on MS medium containing a combination of 0.5 mg L^?1 6-BA with 0.6 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-d). Optimal shoots differentiated from callus were obtained on MS medium supplemented with 5.0mg L^?1 6-BA and 0.9 mg L^?1 NAA. In vitro rooting was achieved on half-strength (1/2) MS medium containing 0.5 mg L^?1 NAA. Rooted plantlets were hardened under control conditions and successfully acclimatized under field conditions.     

Micropropagation of Anogeissus rotundifolia Blatt,and Hallb.-An Endemic and Rare Tree of the Thar Desert

Abstract

Multiple shoots and plantlets were developed in vitro from cotyledonary nodal segments of in vitro raised seedlings of Anogeissus rotundifolia (syn. A. sericea var. nummularia)-a rare and endemic tree species of the Thar Desert. About 15-20 shoots differentiated from a single cotyledonary node within four weeks on Mu-rashige and Skoog's (MS) basal medium containing 0.1 mg l^?1 indole-3-acetic acid (IAA) + 2.0 mg l^?1 6-benzylaminopurine (BAP) + additives (25 mg l^?1 each of adenine sulphate, L-arginine, and citric acid, and 50 mg l^?1 of ascorbic acid) at 26 ± 2°C temperature and 36 μmol m^?2 s^?1 photon flux density with a 12 h/day photoperiod. The shoots produced in vitro were further multiplied by subculturing on fresh medium. The original cotyledonary nodal segment was repeatedly transferred (5 to 6 times) onto fresh medium containing 1.0 mg l ^?1 BAP + 0.1 mg I ^?1 IAA + additives to yield fresh crops of multiple shoots. These shoots were rooted on half-strength MS medium supplemented with 0.1 mg l^?1 indole-3-butyric acid (IBA). Plantlets were transferred to pots containing sand-dune soil and ver-miculite it the ratio of 4:1 (v/v) and hardened in a growth chamber for two weeks and finally transferred to a greenhouse. From a single cotyledonary node about 1500 plantlets could be developed within four months. The method developed is useful for mass multiplication and for the conservation of germplasm of Anogeissus rotundifolia.     

Regeneration of <Emphasis Type="Italic">Melia volkensii</Emphasis> Gürke (Meliaceae) through direct somatic embryogenesis

Experiments were conducted to study plant regeneration through direct somatic embryogenesis using mature zygotic embryo and cotyledonary explants from seeds of Melia volkensii stored for <3 and >12 months. Explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP, NAA and 2,4-D (0.5, 1.0 and 2.0 mg l⁻¹) alone, and BAP (0.5, 1.0, 2.0 and 4.0 mg l⁻¹) in combination with 2,4-D or NAA (0.2 and 0.5 mg l⁻¹). After 4 weeks in culture, up to 60% of cotyledonary explants from the seeds stored for <3 months produced direct somatic embryos on BAP (0.5–4.0 mg l⁻¹) in combination with 2,4-D (0.2 mg l⁻¹). The number of somatic embryos ranged from 5 to 14 per explant in BAP (0.5 mg l⁻¹) and 2,4-D (0.2 mg l⁻¹) combination. Only 20% of cotyledonary explants from seeds stored for >12 months produced somatic embryos. Mature zygotic embryos failed to produce any somatic embryos. Subcultures of somatic embryos from cotyledonary explants of seeds stored for <3 months formed clusters of shootlets on semi solid MS and 1/2 MS media. After 6 weeks of subculture on multiplication MS media augmented with BAP (0.5 mg l⁻¹) and IAA (0.2 mg l⁻¹), 70% of the shoot tips formed 4–7 shoots per explant. Up to 33% of the multiplied shoots were rooted in MS medium supplemented with 2.0 mg l⁻¹ IBA. Plantlets developed normally into seedlings in the greenhouse.     

Clonal propagation ofGmelina arborea Roxb. byIn vitro culture

In vitro propagation technique ofGmelina arborea multipurpose and a fast growing tree species was studied. Nodal segment including axillary bud was used as a explant. They were cultured on MS media containing various concentrations (0–10 mg/l) of BAP alone or in combination with 0.002 mg/l of IBA. Nodal segments showed axillary bud proliferation in almost all media tested. MS media containing 0.22 mg/l of BAP alone and 2 mg/l of BAP in combination with 0.002 mg/l of IBA were effective for inducing multiple shoots and shoot elongation. MS medium supplemented with 0.02 mg/l of NAA and 1 mg/l of IBA gave the best result for rooting. The regenerated plantlets were potted and acclimatized successfully in a growth chamber and then moved to the green house. Adventitious shoots production from stem explants that were taken from regenerated plantletin vitro was also discussed. Stem segments were tested for their morphogenetic potential on MS media with various combinations and concentrations of BAP, zeatin and TDZ. Successfull result was obtained on MS media supplemented with 2 mg/l of BAP and 1 mg/l of zeatin or supplemented with 0.5 mg/l of BAP and 0.5 mg/l of TDZ. The shoots obtained on MS media containing 2 mg/l of BAP and 1 mg/l of zeatin rooted on MS media containing 0.02 mg/l of NAA and 1 mg/l of IBA, and plantlets were successfully obtained. A part of this paper was presented at the 109th Annual Meeting of the Japanese Forest Society (1998).     

Using synergistic exogenous phytohormones to enhance somatic embryogenesis from leaf explants of a Eucalyptus grandis clone

Somatic embryogenesis (SE) in Eucalyptus spp. has been limited to germinated seeds, flowers, lignotubers or zygotic embryos. The low yield of somatic embryos from leaf explants has hampered progress, even though leaves offer a more viable source of clonal explants from superior selected genotypes. It was hypothesised that SE from leaf explants could be enhanced through pairing of synergistic exogenous plant growth regulators, such as natural auxins with natural cytokinins. Callus and embryo induction using 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (NAA), indole acetic acid (IAA), and indole butyric acid (IBA), each at either 1.0 or 3.0?mg L^?1, indicated that IAA and IBA favoured significantly higher numbers of embryos compared with 2,4-D or NAA. Hence, IAA and IBA were used for subsequent experiments, combining them (at 1.0?mg L^?1) with either the synthetic cytokinin, kinetin, or the natural cytokinin, trans-zeatin, both at 0.1?mg L?1. The combination of trans-zeatin and either IAA or IBA resulted in a significant increase in SE (e.g. 86 ± 17.2% and 23 ± 3.2% for IAA with trans-zeatin and kinetin, respectively), compared with kinetin, or with these auxins alone. Embryo maturation and plantlet regeneration was highest in those calli that were induced with IAA and trans-zeatin, indicating that maturation was dependent on auxin depletion, based on the stability of the analogue used for induction. For the E. grandis clone under study, the use of synergistic plant growth regulators significantly enhanced SE from leaf explants, thus presenting the opportunity to benefit from the advantages that SE offers over conventional organogenesis.     

Micropropagation of Selected Genotype of Aloe vera L.—An Ancient Plant for Modern Industry

Aloe vera Linn. (Syn. Aloe barbadensis Mill; Gwar-patha in Hindi) belongs to family Liliaceae. The plant, for its medicinal properties, has commercial value. Some of the genotypes of Aloe vera are consumed as a vegetable and processed to make curry and other edible products. We report here on the development of an efficient method for rapid clonal propagation by shoot proliferation from axillary meristem(s) of selected germplasm of Aloe vera. Explants were pretreated with 0.1% aqueous solution of both streptomycin and bavistin separately, each for 15 min. These were surface sterilized with 0.1% aqueous solution of mercuric chloride (HgCl₂) for 4–5 min and washed several times with autoclaved water. These were kept in a chilled, sterile antioxidant (200.0 mg L^?1 of ascorbic acid, 50.0 mg L^?1 of citric acid, and 25.0 mg L^?1 of polyvinylpyrrolidone; PVP) solution and cultured on semi-solid Murashige and Skoog's (MS) medium. The bud explants produced multiple (10.3 ± 0.675/explant) shoots on MS medium containing 13.32 μM of 6-Benzylaminopurine (BAP) and 100.0 mg L^?1 of ascorbic acid, 50.0 mg L^?1 each of citric acid and PVP, with 25.0 mg L^?1 each of arginine and adenine sulphate as additives. The shoots were further multiplied by (a) repeated transfer to fresh MS medium with additives + 13.32 μM BAP, and (b) subculturing on MS medium with a lower (4.44 μM) concentration of BAP. On MS medium containing 4.44 μM of BAP and additives, a maximum number (27.8 ± 0.63) of shoots were produced. In liquid MS medium with 4.44 μM of BAP, the rate of shoot multiplication increased and the vigor of the shoots improved. One hundred percent of the cloned shoots rooted under in vitro conditions on hormone-free half-strength MS salts containing 200.0 mg L^?1 of activated charcoal at 32 ± 2°C. The cloned shoots treated with 2.46 mM of indole-3-butyric acid (IBA) or 2.473 mM of β-naphthoxyacetic acid (NOA) for 5 min rooted under ex vitro conditions in the greenhouse. The rooted plants were hardened in the greenhouse and stored under an agro-net house. The cloned plants were transferred under different field conditions at various sites in Western Rajasthan. These plants grew normally. The higher rate of shoot multiplication and easier approach of direct rooting and hardening make this method superior to the methods previously reported on cloning/tissue culture of Aloe species. From a single shoot bud, approximately 5000 plants can be produced within 180 days.     

Plantlet regeneration from leaf protoplasts ofPopulus euphratica

Protoplasts were isolated from the leaves of sterile plants ofPopulus euphratica Oliv. by using 1% Cellulase “Onozuka” RS and 0.25% Pectolyase Y-23 in 0.6m of mannitol solution. Protoplasts were cultured in modified Murashige and Skoog's (MS) medium which contained no ammonium ions but was supplemented with BAP (6-benzylaminopurine), 2,4-D (2,4- dichlorophenoxy-acetic acid), and 1% sucrose at the cell density of 9×10⁴/ml. Cell divisions occurred in every culture medium, especially in the medium containing 0.5 mg/l of BAP and 0.1 mg/l of 2,4-D, in which callus was successfully induced by successive culture through cell cluster formation. Shoots were regenerated from the callus, and their growth was enhanced on 1/2 MS medium containing 0.8 mg/l of BAP. Finally, shoots were rooted and plantlets were regenerated on 1/2 MS medium without a hormone. A part of this paper was presented at the 106th Annual Meeting of the Jpn. For. Soc. (1995).     

An Efficient Micropropagation Protocol for High-Frequency Plantlet Regeneration from Liquid Culture of Nodal Tissues in a Medicinal Plant,Turnera ulmifolia L.

A highly efficient, stable, and cost-effective micropropagation protocol for the conservation of a medicinal plant Turnera ulmifolia L. was established from nodal tissues via multiple axillary shoot proliferation on using Murashige and Skoog’s (MS) liquid nutrient medium. To begin with, nodal explants were placed on agar gelled medium amended with 2.0 mg L^?1 6-benzylaminopurine (BAP) and 0.1 mg L^?1 indole-3 acetic acid (IAA) for shoot induction. Subsequently, elongation of regenerated shoots could be possible on liquid MS medium supplemented with 0.5 mg L^?1 BAP and Kin (kinetin) each along with 0.1 mg L^?1 IAA where high frequency of regeneration in terms of number of shoots (47.2 shoots/explant) was achieved. Furthermore, long and healthy shoots (4?5 cm in length) were rooted on agar gelled half-strength of MS medium supplemented with 2.0 mg L^?1 indole-3 butyric acid (IBA). Finally, in vitro regenerated plantlets were gradually acclimatized in the greenhouse and transferred to the field successfully.     

Effects of plant growth regulators,carbon sources and pH values on callus induction in Aquilaria malaccensis leaf explants and characteristics of the resultant calli

The endangered tropical tree, Aquilaria malaccensis, produces agarwood for use in fragrance and medicines. Efforts are currently underway to produce valuable agarwood compoundsn tissue culture. The purpose of this study was to develop an optimal growth medium, specifically, the best hormone combination for callus suspension culture. Using nursery-grown A. malaccensis, sterilized leaf explants were first incubated on basic Murashige and Skoog(MS) gel medium containing 15g/L sucrose and at pH 5.7. Different auxin types including 1-naphthaleneacetic acid(NAA), 2,4-dichlorophenoxyacetic acid(2,4-D), and indole-3-butyric acid(IBA), were tested at various concentrations(0.55, 1.1 and 1.65 μM) using the basic medium. Leaf explants were incubated for 30 days in the dark. Callus induced by 1.1 μM NAA had the highest biomass dry weight(DW) of 17.3 mg; however the callus was of a compact type. This auxin concentration was then combined with either 6-benzylaminopurine(BAP) or kinetin at 0.55, 1.1, 2.2 or 3.3 μM to induce growth of friable callus. The 1.1μM NAA + 2.2μM BAP combination produced friable callus with the highest biomass(93.3mg DW). When testing the different carbon sources and pHs, sucrose at 15g/L and pH at 5.7 yielded highest biomasses at 87.7mg and 83 mg DW, respectively. Microscopic observations revealed the arrangement of the friable cells as loosely packed with relatively large cells, while for the compact callus, the cells were small and densely packed. We concluded that MS medium containing 15 g/L sucrose, 1.1 μM NAA + 2.2 μM BAP hormone combination, and a pH of 5.7 was highly effective for inducing friable callus from leaf explants of A. malaccensis for the purpose of establishing cell suspension culture.     

Analysis of Spacing for Spotted Gum Plantations for Maximizing Merchantable Logs' Volume in Southeast Queensland,Australia

Spotted gum (Corymbia citriodora subspecies Variegata) has the potential to be the major hardwood species for large-scale plantations in Southeast Queensland, Australia, but production research is limited due to the lack of age of research plots. Optimal spacing is a major subject of concern. Based on time series data from a spotted gum experiment site, growth performance was analyzed for five spacing levels: 11.3 m?×?11.3 m (78 stems ha^?1), 7.4 m?×?7.4 m (182 stems ha^?1), 5.4 m?×?5.4 m (343 stems ha^?1), 3.6 m?×?3.6 m (771 stems ha^?1), and 2.9 m?×?2.9 m (1189 stems ha^?1). The major objective was assumed to be to maximize total merchantable log volume. A growth model was produced, and the mean diameter at breast height (DBH) and total merchantable log volume for each spacing level at a range of harvesting ages were estimated. From the analysis, the spacing level of 5.4 m?×?5.4 m was found to be optimal for maximizing merchantable log volume to a 10-cm small-end diameter. Further analysis of mean DBH, height, and volume of the largest 200 and 250 trees from this spacing level indicates that merchantable log volume could be maximized by retaining the 250 largest trees ha^?1. The total financial revenue from the best spacing level in 25 and 30 yr are predicted to be Australian dollars (A$) 13,637 and A$17,779 ha^?1, respectively. If full rotation data could be obtained, more reliable models could be produced, and a more accurate financial estimate could be made.     

Effect of tree spacing on growth and wood density of 38-year-old Cariniana legalis trees in Brazil

Average wood density of 38-year-old Cariniana legalis (Mart.) Kuntze, a Brazilian native forest species, was found to increase with faster growth and lower stocking, while decreasing from pith to bark. A complete randomised block design was planted with five blocks. Ten trees were harvested in each of three spacing treatments. We hypothesised that the stand stemwood production would not significantly differ depending on tree spacing. However, tree growth would be higher in the wider spacing and wood density would be higher in the narrower spacing. The diameter growth of trees was higher at 3 m × 2.5 m than at 3 m × 2 m and 3 m × 1.5 m. Nevertheless, this higher individual tree growth at 3 m × 2.5 m did not compensate for the greater tree stock density at 3 m × 1.5 m with stand stemwood production at 38 years of 530 m3 ha^?1 and 649 m³ ha^?1, respectively. These results suggest that C. legalis, which can produce up to 17 m³ ha^?1 y^?1 of medium-to high-density timber – about 800 kg m^?3 – is a promising native species for forest plantations in Brazil.     

Establishment of an in vitro plant regeneration protocol for Casuarina cunninghamiana Miq. via indirect organogenesis

An in vitro plant regeneration protocol via indirect organogenesis from morphogenetic callus was established for Casuarina cunninghamiana Miq. Effects of plant growth regulator NAA (naphthaleneacetic acid) and BAP (6-benzylaminopurine), sucrose and AgNO₃ on callus induction, adventitious bud differentiation and shoot development were examined. Explants used were epicotyl fragments from 45-day-old seedlings. The largest callus (4.29 mm in diameter) was obtained after 1 month on a basic culture medium consisting of Murashige and Skoog ? macro- and full strength micro- elements, Nitsch and Nitsch vitamins, supplemented with 0.54 μM NAA, 3.30 μM BAP, and 30 g L⁻¹ sucrose. The calli were subcultured in the same medium above for 2 months. They were then cultured for another 2 months for adventitious bud differentiation and shoot development. The highest mean adventitious bud differentiation, number of shoots formed per callus and number of shoots ≥2 cm long per callus (47.50%, 27.38 and 4.75, respectively) were achieved on the above medium modified with NAA at 0.27 μM and supplemented with AgNO₃ 1 mg L⁻¹. Shoots were successfully rooted without plant growth regulator and the rooted plantlets survived and grew normally. This protocol for in vitro plant regeneration provides a tool not only for vegetative propagation but also for plant genetic transformation and gene function studies of C. cunninghamiana.     

Micropropagation of Leptadenia pyrotechnica (Forsk.) Decne: A Multipurpose Plant of an Arid Environment

Leptadenia pyrotechnica (Forsk.) Decne belongs to the family Asclepiadaceae. It is commonly known as Kheemp in India. L. pyrotechnica is an important component of an arid ecosystem and source of fiber, forage, and medicines. We report here in vitro propagation protocol of L. pyrotechnica. Cotyledonary nodes of in vitro raised seedlings were used as an explant for multiplication of shoots. Shoots were multiplied on Modified Murashige and Skoog's (MMS) medium containing 1.33 μM BAP and additives (50 mg L^?1 ascorbic acid and 25 mg L^?1 each of citric acid, arginine, and adenine). Cultures were maintained at 30 ± 2°C temperature, 50–60 μmol m^?2 s^?1 SFP, 12 hr day^?1 photoperiod, and 60% relative humidity (RH). In vitro multiplied shoots were rooted in vitro on half-strength MS medium containing 2.46 μM IBA and 100 mg L^?1 activated charcoal. Shoots were rooted ex vitro using 2460 μM IBA pretreatment for 15 min. Plantlets were hardened in a greenhouse, and survived on a mixture of sand, garden soil, and organic manure in 3:1:1 ratio in polybags in natural habitats.     

Development of an efficient protocol for plant regeneration from nodal explants of recalcitrant bamboo (Bambusa arundinacea Retz. Willd) and assessment of genetic fidelity by DNA markers

The present study describes an efficient method for in vitro plant regeneration in B. arundinacea through axillary shoot bud proliferation. Nodal explants were excised, cultured on MS medium containing different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN) (0.5–5.0 mg l^?1) alone and/or in combinations with KIN/BAP (0.5 mg l^?1). The highest frequency (91.5 %) of multiple shoot bud induction with maximum number of shoots (85 shoots/explant) was noticed on MS medium + 3.0 mg l^?1 BAP + 0.5 mg l^?1 KIN. The regenerated multiple shoots were elongated on MS medium + 4.0 mg l^?1 KIN + 2.0 mg l^?1 gibberellic acid (GA₃) with maximum shoot length (4.9 cm). The elongated shoots were transferred to MS medium containing indole-3 butyric acid (IBA; 0.5–5.0 mg l^?1) alone and/or in combination with 0.5 mg l^?1 KIN and BAP. Highest frequency of rooting (75 %) was obtained on half-strength MS medium + 2.0 mg l^?1 IBA + 0.5 mg l^?1 KIN. After hardening, the plantlets were shifted to the green house and subsequently established in the field conditions with 90 % survival rate. random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of the regenerants. RAPD profiles generated from the regenerated plants were found to be monomorphic, similar to the control. Results confirmed that the regenerated plants were true-to-type in nature and the developed micropropagation protocol could be used for large scale plant production of B. arundinacea.     

Micropropagation and callus culture of <Emphasis Type="Italic">Saussurea laniceps</Emphasis>, an alpine medicinal plant

Cottonhead windhairdaisy (Saussurea laniceps Hand.-Mazz.) is one of the most famous and important medicinal herbs in China. Illegal collection from wild populations is increasingly threatening the present environment of S. laniceps. Establishment of an efficient method for micropropagation is the best way to change its endangered situation. When mature seeds of S. laniceps were cultured on hormone-free MS medium, plantlets were formed from germinated seeds in 7–10 d. Then 0.5 cm × 0.5 cm leaf explants were transplanted to MS medium supplemented with 1-naphthalene-acetic acid (NAA)/2,4-D and benzyladenine (BA)/KT and callus was achieved 10 d after transfer. Shoot bud regeneration occurred from callus cultured on MS medium supplemented with different growth regulators 20 d after culturing. The regeneration percentages varied with the different components of plant growth regulators. The percent regeneration from callus pretreated at low temperature of 5°C increased significantly compared with those incubated at 23/20°C directly. Optimal regeneration was observed with explants on media supplemented with 1.5 mg&#8226;L^–1 BA plus 0.2 mg&#8226;L^-1 NAA. In the presence of 0.2 mg&#8226;L^–1 NAA in half-strength MS, 78% of the shoots formed roots. Plantlets from explants showed 63% survival after acclimatization.     

Micropropagation of Vegetable Rennet (Withania coagulans [Stocks] Dunal)—A Critically Endangered Medicinal Plant

Withania coagulans (Stocks) Dunal (Solanaceae), popularly called vegetable rennet, is a critically endangered and highly valued medicinal plant. Overexploitation and reproductive failure forced the plant species toward the verge of complete extinction. We describe here the development of a simple, rapid, and cost effective in vitro micropropagation system for W. coagulans for mass-scale production of true-to-type plantlets using nodal shoot segments. Exactly 95.5 ± 0.34% explants responded within 8–10 days (d) and produced multiple shoot buds (4.1 ± 0.10 shoots of 2.95 ± 0.15 cm length) on 0.8% agar-gelled Murashige and Skoog's (MS) basal medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), and additives (100 mg L^?1 L-ascorbic acid, 25 mg L^?1 each citric acid, adenine sulphate, and L-arginine). The shoots in cultures were multiplied by repeated transfer on MS medium with 4.44 μM BAP, 0.57 μM IAA, and additives. Further cultures were multiplied on a large-scale through the subculturing of shoot clumps differentiated in vitro, on MS medium supplemented with 1.11 μM BAP, 0.57 μM IAA, and additives. Maximum number (19.1 ± 0.28) of healthy (6.15 ± 0.25 cm) and viable shoots differentiated on this medium. The microshoots were rooted both in vitro and ex vitro. Exactly 67.3 ± 1.01% microshoots rooted in vitro within 25–30 d on agar-gelled half-strength MS salts supplemented with 29.52 μM indole-3-butyric acid (IBA) and 200 mg L^?1 of activated charcoal (AC). Alternatively, 73.8 ± 0.65% cloned shoots rooted on sterile soilrite (soilless compost and soil conditioner) under ex vitro conditions after pulse treatment with 2.46 mM IBA for 300 s. The clones of W. coagulans were hardened in a greenhouse within 40–45 d by slow and gradual exposure of plantlets from high relative humidity (RH; 70–80%) and low (26 ± 2°C) temperature to low RH (40–50%) and high (34 ± 2°C) temperature. The hardened plantlets were transferred to soil and stored in agro-net house with more than 90% survival rate. Replacement of pure and laboratory grade sucrose with commercial grade sugar, use of less expensive commercial grade agar-agar in culture medium, higher rate of shoot proliferation, single step ex vitro rooting, and hardening of plantlets in the greenhouse are advantageous features of the protocol. The micropropagation protocol defined here is reproducible, easy to follow, and would be helpful in large-scale restoration programs through true-to-type mass-multiplication of W. coagulans.     

Early growth of Eucalyptus camaldulensis under agroforestry conditions at Mafiga,Morogoro, Tanzania

The growth of Eucalyptus camaldulensis clean weeded, spot weeded and intercropped with maize and beans was studied. At 4 m × 4 m and 5 m × 5 m spacings trees were significantly shorter after 15 months under a conventional spot weeding regime than with clean weeding or intercropping with beans. A satisfactory maize yield (683 kg ha^?1) was recorded from plots with trees spaced at 5 m × 5 m. Plots where trees were spaced at 4 m × 4 m and 3 m × 3 m gave significantly lower yields (444 kg ha^?1 and 283 kg ha^?1, respectively).     

Efforts Toward Improving Maize Yields on Smallholder Farms in Uasin Gishu County,Kenya, through Site-specific,Soil-testing-based Fertiliser Recommendations: A Transdisciplinary Approach

This study evaluated the effects of site-specific, soil-testing-based fertiliser recommendations on maize yields using the transdisciplinary (TD) process. The TD process utilizes knowledge from science and practice. Farmers, extension officers, local financial institutions, and other practitioners collaborated with local scientists from the University of Eldoret in the process of financing, purchasing, and applying fertilisers in adequate amounts and composition. A total of 144 farmers participated in the study, which lasted for two seasons. The data sampling was based on a randomized 2?×?3?×?4?×?2 factorial complete block design, including the following factors: TD (non-participation vs participation in the TD process); ST (soil testing in the following categories: fertiliser application with no soil testing, fertiliser application following government recommendations, and application of site-specific, soil-testing-based fertiliser recommendations), and location (Kapyemit, Kipsomba, Ng’enyilel, and Ziwa). The “no soil testing” (ST₁) category refers to farmers’ own practices at an average fertilisation of about 60?kg?N?ha^?1 and 15?kg?P?ha^?1. The government recommendation (ST₂) calls for 75?kg?N?ha^?1, 25?kg?P?ha^?1, and 6?t?ha^?1 manure, and site-specific fertiliser recommendations (ST₃) were based on actual soil-testing results; generally, this resulted in the recommendation of 90?kg?N?ha^?1, 30?kg?P?ha^?1, 25 kg K ha^?1, 2?t?ha^?1 lime, and 1?t?ha^?1 manure. Highly significant effects were seen where farmers participated in the TD process (TD) for soil testing (ST). The farmers’ yields in Uasin Gishu County of 4.5?t?ha^?1 increased by approximately 1.5?t?ha^?1 based on site-specific, soil-testing fertilisation recommendations and by approximately 1.0?t?ha^?1 based on participation in the transdisciplinary process. However, as indicated by a significant interaction of the variables ST and TD—and while there is a significant main effect of participating in a TD process—the latter increase occurs only if site-specific, soil-testing-based recommendations can be used in the transdisciplinary process with farmers.     

Long-term effect of wood and peat ash on the growth of Pinus sylvestris on drained peatland

The effect of ash fertilization on height growth and volume production of Scots pine (Pinus sylvestris L.) was studied on oligotroph peatland in southeast Norway. In the year 1944, plots 15 m × 15 m size were fertilized with 0, 4, 7, and 10 tons ha^?1 of wood or peat ash. The area was treeless, but a satisfactory number of pine seedlings were present. All measurements were confined to the central inner plot, 10 m × 10 m area. Most plots were re-fertilized with 10 tons of wood ash ha^?1 in the year 1993. Wood ash had higher content of nutrients, and generally, it had greater growth enhancement effect than peat ash. When the amount of ash was increased, volume production significantly increased for the age period 38–50 years and the total production at age 50 years. The mean annual increment during the first 50 years was about 6 m³ ha^?1 for the plots applied with 10 tons of ash ha^?1. Trees on plots fertilized with 7 or 10 tons in 1944 and replenished with 10 tons ha^?1 at age 50 years (1993) had a mean annual increment of 14 m³ ha^?1 for the stand age period 51–68 years. Over time some tree roots from control plots and plots fertilized with 4 tons ha^?1 have captured nutrients from richer plots. Such effect is to a smaller extent relevant for treatment 7 tons. It is concluded that the content of mineral nutrients of wood and peat ash makes these ashes well suited as fertilizers on peatland.