Promoting root induction and growth of in vitro macadamia (<Emphasis Type="Italic">Macadamia tetraphylla</Emphasis> L. ‘Keaau’) plantlets using CO<Subscript>2</Subscript>-enriched photoautotrophic conditions

In this study, a rooting protocol was developed for macadamia plantlets with healthy roots and enhanced growth performance, along with enhanced photosynthetic capability. In vitro-grown shoots rooted in vented vessels containing vermiculite as the supporting material exhibited 100% frequency of root induction, whereas when shoots were grown in non-vented vessels containing a solidified Murashige and Skoog (MS) medium, the frequency of root induction was less than 30%. The formation of root with callus, hyperhydricity, and leaf necrosis was observed in this photomixotrophic closed system. The modification of the vented photoautotrophic system with different concentrations of CO₂ and sucrose were investigated using vermiculite as the supporter. The number of roots, root length, root surface area, fresh weight, and dry weight were significantly higher in plantlets grown in CO₂-enriched (1,000 μmol CO₂ mol⁻¹) photoautotrophic conditions. The water content in both root and shoot tissues of plantlets cultured under photoautotrophic conditions was maximized. In addition, shoot and leaf performances were enhanced in plantlets cultured under CO₂-enriched photoautotrophic conditions. The supplementation of sucrose (29–88 mM) to culture media in both ambient and elevated CO₂ conditions affected a reduction in the shoot and root performance of in vitro plantlets. Chlorophyll a, chlorophyll b, and total carotenoids in the leaf tissues of plantlets acclimatized in CO₂-enriched photoautotrophic conditions were enriched, leading to increasing photosynthetic abilities, including chlorophyll fluorescence and net photosynthetic rate. From this investigation, a root induction protocol was established and the production of healthy macadamia plantlets was successfully implemented using CO₂-enriched photoautotrophic conditions.     

Interactions of culture vessels, media volume, culture density, and carbon dioxide levels on lettuce and spearmint shoot growth in vitro

 The influence of culture chamber capacity, medium volume and culture density on the growth yields of lettuce (Lactuca sativa L.) and spearmint (Mentha spicata L.) shoots were determined in an environment containing either 350 or 10,000 μmol mol^–1 CO₂ after 8 weeks of incubation. High positive correlations occurred between the culture vessel capacity and spearmint fresh weight, leaf number, root number, and shoot number. Similarly, high positive correlations occurred between culture vessel capacity and lettuce fresh weight, leaf number, and root number. Higher fresh weights, leaf numbers, and root numbers were obtained from lettuce and spearmint shoots when cultured in 1-quart Mason jars containing 100- or 150-ml aliquots of medium compared to jars containing 25- or 50-ml aliquots of medium within an environment containing either 350 or 10,000 μmol mol^–1 CO₂. High culture density decreased growth yields, and this phenomenon could only be slightly off-set by the employment of an elevated CO₂ environment or larger culture vessels. Received: 22 December 1998 / Revision received: 2 July 1999 / Accepted: 12 July 1999     

Testing a new hypothesis: plant vigor and phylloxera distribution on wild grape in Arizona

Summary Longer, meaning more vigorous, shoots of a wild grape clone (Vitis arizonica) were more susceptible to attack by second and third generations of leaf-galling grape phylloxera,Daktulopsphaira vitifoliae, as the growing season progressed. Although there was no significant difference in mean shoot length between attacked and unattacked shoots within a clone at the beginning of shoot elongation, attacked shoots were significantly longer than unattacked shoots when elongation had ceased (P<0.01). Also, long attacked shoots had a significantly greater population of phylloxera galls than short attacked shoots (P<0.01) as the season progressed. The phylloxera population on long shoots increased rapidly while the population on short shoots remained the same. Longer shoots also produced significantly more axillary shoots than shorter shoots as the season progressed (P<0.001), and the number of axillary shoots accounted for 66 percent of the variance in number of attacked leaves on a shoot. Experimental evidence showed that there was a significantly greater percentage of available leaves attacked on long shoots than on short shoots (P<0.05) and the leaves on long shoots generally had a greater number of galls per leaf. The relationship between shoot length and probability of attack was also tested by comparing shoots lengths of 10 attacked clones and 10 unattacked clones at a second location. Mean shoot lengths of attacked clones were significantly longer than mean shoot lengths of unattacked clones (P<0.05), and mean shoot lengths of attacked shoots within a clone were significantly longer than unattacked shoots (P<0.001). Longer shoot length accounted for 81 percent of the variance in probability of attack. The reason for this pattern of attack was that long shoots produced newly expanding leaves over a longer time during the growing season and multivoltine phylloxera require undifferentiated tissue to initiate gall formation. Patterns of attack within a shoot were characterized by an uneven distribution of galls among leaves. This was due to development time between generations and the current availability of undifferentiated tissue at times of colonization. This study supports the hypothesis that some herbivore species are favored more by vigorous plants than by stressed plants.     

CO2-Enrichment and photosynthetic photon flux affect the growth of in vitro-cultured apple plantlets

Micro-cuttings (shoots with two small leaves) of cultivar M9 apple were cultured in-vitro for 40 d under CO₂-enriched and non-enriched (i.e., ambient air) conditions, and at a PPF of 40 or 100 μmol m^-2 s^-1 Afterward, shoot length, number of leaves, leaf area, chlorophyll content, shoot and root fresh weights, and % survival were recorded. Those plant-lets grown under CO₂- and PPF-enriched treatments were healthy and vigorous, and showed higher values for their growth parameters. In contrast, those grown without supplemental CO₂ or PPF often showed hyperhydricity. We also demonstrated that CO₂ enrichment and a relatively high PPF during in-vitro culture promoted normal photosynthesis and growth after ex-vitro transplantation.     

Growth of In vitro banana (Musa SPP.) shoots under photomixotrophic and photoautotrophic conditions

Summary In vitro banana (Musa spp.) shoots were cultured under photomixotrophic (30 gl⁻¹ sucrose and 0.2 h⁻¹ number of air exchanges of culture vessels) and photoautotrophic (0 gl⁻¹ sucrose and 3.9 h⁻¹ number of air exchanges) conditions for 28 d in 370 cm³ Magenta boxes (GA7-type) containing 70 ml of half-strength Murashige and Skoog (MS) medium with 22.2 μM N⁶-benzyladenine (BA). The effects of varying CO₂ concentration (475 or 1340 μmol mol⁻¹) and light intensity (photosynthetic photon flux (PPF) of 100 or 200 μmol m⁻² s⁻¹) were investigated. Fresh and dry weights of banana shoots grown photomixotrophically were significantly greater on day 28 than those grown photoautotrophically. Photoautorophic shoots had a larger number of unfolded leaves and greater leaf area than photomixotrophic plants by days 14 and 28, regardless of CO₂ concentration. The shoot fresh and dry weights on day 14 in photoautotrophic conditions were significantly greater at PPF of 200 μmol m⁻² s⁻¹ than at 100 μmol m⁻² s⁻¹. The increase in net photosynthetic rate of photoautotrophic banana shoots was significant compared with photomixotrophic shoots. The multiplication ratio of in vitro banana shoots grown photoautotrophically in a 28-d culture period was the greatest at 100 μmol m⁻² s⁻¹ PPF and 475 μmol mol⁻¹ CO₂.     

In vitro Growth and Shoot Multiplication of Achras zapota in a Controlled Carbon Dioxide Environment

The culture vessels with multiplying shoots of Achras zapota L. on Schenk and Hildebrandt (SH) medium containing 8.88 M 6-benzylaminopurine (BAP) with or without sucrose were kept under varied CO₂ concentrations ranging from 0.6 to 40.0 g m^–3 using different concentrations of sodium bicarbonate (NaHCO₃), sodium carbonate (Na₂CO₃), potassium bicarbonate (KHCO₃), and potassium carbonate (K₂CO₃) in small acrylic chambers. Complete absence of carbon source caused death of shoots within 20 d. Under elevated concentrations of CO₂ (10.0 and 40.0 g m^–3) the shoots grew photoautotrophically on sucrose-free medium. The growth of cultures was better at 40.0 g (CO₂) m^–3 than on 3.0 % sucrose under ambient air of growth room. However, the best response was obtained at 10.0 g (CO₂) m^–3 and 3.0 % sucrose where maximum number of shoots, shoot length, fresh and dry mass, total number of leaves and leaf area was observed.     

In vitro growth and shoot multiplication of Wrightia tomentosa Roem et Schult in a controlled carbon dioxide environment

In vitro growth and multiplication of shoots of a woody tree species Wrightia tomentosa in a controlled carbon dioxide environment was studied. The cultures were grown on BA supplemented MS medium with or without 3% sucrose. A range of CO₂ concentrations (0.0, 0.6, 10.0 and 40.0 g m^–3) was controlled in small chambers by using solutions of NaHCO₃, Na₂CO₃, KHCO₃ and K₂CO₃. To obtain a CO₂-free environment, a saturated solution of KOH was kept in the chambers. It was concluded that the growing shoot cultures required either sucrose in the medium as a carbon source or an ambient CO₂ environment. Complete absence of a carbon source caused severe browning of the shoots and death within 30 days. The cultures grew better with 10.0 g m^–3 carbon dioxide in the environment than with 3.0% sucrose in the medium. With both CO₂ and sucrose being available, the best response was obtained at 0.6 g m^–3 CO₂ in the chamber. At this concentration the rate of shoot multiplication was nearly double the standard rate obtained when exposed to the natural CO₂ level and sucrose-supplemented medium. Total fresh and dry weight, leaf number and area per cluster also showed the best response under this condition.     

A rapid in vitro propagation of red sanders (<Emphasis Type="Italic">Pterocarpus santalinus</Emphasis> L.) using shoot tip explants

An efficient protocol has been developed for the in vitro propagation of Pterocarpus santalinus L. using shoot tip explants which is a valuable woody medicinal plant. Various parts of this plant are pharmaceutically used for the treatment of different diseases. Multiple shoots were induced from shoot tip explants derived from 20 days old in vivo germinated seedlings on 1:1 ratio of sand and soil after treating with gibberellic acid (GA₃). The highest frequency for shoot regeneration (83.3%) with maximum number of shoot buds (11) per explant was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l of 6-benzylaminopurine (BAP) along with 0.1 mg/l of thidiazuron (TDZ) after 45 days of culture. A proliferating shoot culture was established by repeatedly subculturing the original shoot tip explants on fresh medium after each harvest of the newly formed shoots. Sixty percent of the shoots produced roots were transferred to rooting medium containing MS salts and 0.1 mg/l indole-3-butyric acid (IBA) after 30 days. About 73.33% of the in vitro raised plantlets were established successfully in earthen pots. Random amplified polymorphic DNA (RAPD)-based DNA fingerprinting profiles were generated for the first time using shoot tip explants of this species and confirmed that there was no genetic variability. This protocol might be helpful for the mass multiplication of P. santalinus in the future.     

Promotion ofin vitro shoot formation from excised roots of silktree (Albizzia julibrissin) by an oxime ether derivative and other ethylene inhibitors

Summary This report describes the regeneration response of excised seedling roots of silktree (Albizzia julibrissin) to added ethylene precursors/generators (1-amino-cyclopropane-1-carboxylic acid [ACC], 2-chloroethylphosphonic acid [CEPA]), biosynthesis inhibitors (aminoethoxyvinylglycine [AVG], an oxime ether derivative [OED={[(ispropylidene)-amino]oxy}-acetic acid-2-(methoxy)-2-oxoethyl ester], CoCl² [Co⁺⁺]), and an ethylene action inhibitor (AgNO₃ [Ag⁺]). When placed on B5 medium, about 50% of the control explants formed shoot buds within 15 days. Addition of ACC or CEPA (1–10 µM) to the culture medium decreased both the percentage of cultures forming shoots and the number of shoots formed per culture. In contrast, AVG and OED (1–10 µM) increased shoot formation to almost 100% and increased the number of shoots formed per culture. Likewise, both Co⁺⁺ and Ag⁺ (1–10 µM) increased shoot regeneration, but the number of shoots produced after 30 days was less than with AVG or OED. The inhibitors of ethylene biosynthesis were partially effective in counteracting the inhibitory effect of ACC on shoot formation. These results suggest that modulation of ethylene biosynthesis and/or action can strongly influence the formation of adventitious shoots from excised roots of silktree.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CEPA 2-chloroethylphosphonic acid - OED oxime ether derivative     

Effect of gibberellin biosynthesis inhibitors on shoot regeneration from hypocotyl explants of Albizzia julibrissin

Summary Hypocotyl explants of Albizzia julibrissin were placed on Gamborg's B5 medium supplemented with various levels of paclobutrazol, uniconazole, prohexadione calcium, or GA₃. Callus formation was evident within one week after placement of the explants on the culture media. Green nodule-like structures protruded from the distal end of the explants within 10 days and developed into shoots within a month. These shoots readily formed adventitious roots when placed on fresh culture medium. All three of the gibberellin biosynthesis inhibitors increased shoot formation compared to the control. The number of shoots per explants was increased 107, 79, and 168% by 0.3–0.4 μM paclobutrazol, uniconazole, and prohexadione calcium, respectively. In contrast to the gibberellin biosynthesis inhibitors, GA₃ decreased shoot formation. These results indicate that modification of gibberellin status can have a strong impact on the number of shoots formed.     

Influence of selected carbohydrates on rhizogenesis of shoots saucer magnolia in vitro

Nodal segements were taken from juvenile shoots of mature 100 year-old trees of saucer magnolia (Magnolia x soulangiana Soul.-Bod.) and cultured on Standardi and Catalano medium supplemented with 1.33 μmol·dm⁻³ BA, 0.54 μmol·dm⁻³ NAA, 58 μmol·dm⁻³ sucrose and 6.0 g·l⁻¹ agar-agar. After 8 weeks, separated shoots were transferred to rooting medium with half-strength macronutrients (basal medium) supplemented with 0.3% activated charcoal and one of carbohydrates: arabinose, cellulose, fructose, galactose, glucose, lactose, mannose, rhamnose, ribose, sorbose, sucrose or xylose at 20 g·dm⁻³ and 7.0 g·dm⁻³ agar-agar. After 13 weeks of culture, shoot number, fresh and dry weight of shoots and roots, total root length and number of roots/per shoot were recorded. Percentages of rooted shoots were calculated. Fructose, mannose and xylose were the most effective carbon source on shoot proliferation followed by sucrose. The rooting response was induced by cellulose and xylose. Arabinose, rhamnose and sorbose inhibited root formation. The number of adventitious roots produced per shoot was stimulated by cellulose and xylose. Total biomass (shoot plus roots) of the plantlets was the highest at fructose and cellulose.     

Somatic embryogenesis and shoot regeneration from transgenic roots of the cherry rootstock Colt (Prunus avium×P. pseudocerasus) mediated by pRi 1855 T-DNA of Agrobacterium rhizogenes

Hairy roots were obtained after inoculation with Agrobacterium rhizogenes strain NCPPB 1855 of the in-vitro-grown shoots of the cherry rootstocks Colt (Prunus avium×P. pseudocerasus) and Mazzard F12/1 (P. avium L.). Not all putatively transgenic roots were able to grow in hormone-free medium. Mazzard F12/1 roots, induced with A. rhizogenes, did not differentiate any shoot or embryo, while both somatic embryos and shoots differentiated from the transgenic roots of Colt in medium containing 1 mg/l 6-benzylaminopurine and 1 mg/l 1-naphthaleneacetic acid. Somatic embryos were capable of secondary embryogenesis, but few developed into whole plants. DNA hybridization showed both a different number of bands and signal intensity in each of the five transgenic shoot clones and embryos examined. In a morphogenetic in vitro test, leaf explants of the transgenic shoot clones showed an increased capacity to differentiate roots, although clones differed in their sensitivity to the hormone ratio. Clones from the transgenic shoots had not only an increased rooting ability when grown in vitro but also exhibited various hairy root phenotypes when cultured in vitro and when transferred into the greenhouse. Received: 12 December 1996 / Revision received: 21 March 1997 / Accepted: 10 May 1997     

Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro

Efficacy of phloroglucinol in promoting growth and development of in vitro-derived shoot tips was studied in six potato (Solanum tuberosum L.) genotypes. Different concentrations of phloroglucinol (0, 0.08, 0.4, 0.8, 1.2 and 1.6 mM) were tested in combination with either 0.1 or 0.2 M sucrose in shoot tip proliferation medium based on MS (Murashige and Skoog, 1962) medium supplemented with 5.8 μM GA₃ (gibberellic acid), 1.1 μM BA (N⁶-benzyladenine) and 8.39 μM D-calcium pantothenate. Phloroglucinol fostered multiple shoot formation, promoted axillary shoot proliferation in terms of shoot tip fresh weight and shoot length, and stimulated root formation on the shoot tips. There was significant phloroglucinol × sucrose interaction for number of shoots developed per shoot tip, shoot tip fresh weight and number of roots induced per shoot tip. The beneficial effect of phloroglucinol on shoot tip survival was conspicuous only in genotypes that showed poor survival in the control proliferation medium. There were significant differences in response between the two sucrose levels with regard to shoot tip fresh weight and number of roots per shoot tip. Phloroglucinol in combination with 0.2 M sucrose induced maximum number of roots per shoot tip. Optimum shoot tip growth was fostered in medium containing 0.8 mM phloroglucinol and 0.2 M sucrose. High frequency multiple shoot formation in this medium ensures a faster rate of potato shoot tip multiplication within a limited time and space. This revised version was published online in June 2006 with corrections to the Cover Date.     

An efficient in vitro plantlet regeneration of <Emphasis Type="Italic">Cryptocoryne wendtii</Emphasis> and <Emphasis Type="Italic">Cryptocoryne becketti</Emphasis> through shoot tip culture

An efficient micropropagation protocol was established for Cryptocoryne wendtii and Cryptocoryne becketti using shoot tips explants. Multiple shoots were induced from shoot tip explants of both species cultured on agar-gelled as well as liquid MS medium supplemented with 0.5 mg/L BA and 0.2 mg/L IBA (proliferation medium). The multiple shoots of both the species formed on agar-gelled as well as liquid medium were vigorously growing with well-developed roots and leaves after 4 weeks of culture. Highest number of multiple shoots was obtained from shoot tip explants of both the species cultured in liquid proliferation medium after 4 weeks of culture. The shoot tip explants of C. wendtii and C. becketti, that were cultured in liquid proliferation medium (2 weeks) followed by culturing on agar-gelled proliferation medium (2 weeks) also produced the multiple shoots. Shoot tips cultured on agar-gelled medium produced the least number of multiple shoots after 4 weeks of culture. Histological study did not show any abnormalities in the leaves of in vitro plantlets of both the species, cultured in agar-gelled and liquid proliferation medium. The leaves of the in vitro plantlets formed normal mesophyll cells and vascular bundles. More than 95% of the acclimatized plantlets grew vigorously without any morphological abnormalities.     

C-isotope composition of CO2 respired by shoots and roots: fractionation during dark respiration?

The CO₂ respired by leaves is ¹³C-enriched relative to leaf biomass and putative respiratory substrates (Ghashghaie et al., Phytochemistry Reviews 2, 145–161, 2003), but how this relates to the ¹³C content of root, or whole plant respiratory CO₂ is unknown. The C isotope composition of respiratory CO₂ (δ_R) from shoots and roots of sunflower (Helianthus annuus L.), alfalfa (Medicago sativa L.), and perennial ryegrass (Lolium perenne L.) growing in a range of conditions was analysed. In all instances plants were grown in controlled environments with CO₂ of constant concentration and δ¹³C. Respiration of roots and shoots of individual plants was measured with an open CO₂ exchange system interfaced with a mass spectrometer. Respiratory CO₂ from shoots was always ¹³C-enriched relative to that of roots. Conversely, shoot biomass was always ¹³C-depleted relative to root biomass. The δ-difference between shoot and root respiratory CO₂ was variable, and negatively correlated with the δ-difference between shoot and root biomass (r² = 0.52, P = 0.023), suggesting isotope effects during biosynthesis. ¹³C discrimination in respiration (R) of shoots, roots and whole plants (e_Shoot, e_Root, e_Plant) was assessed as e = (δ_Substrate − δ_R)/(1 + δ_R/1000), where root and shoot substrate is defined as imported C, and plant substrate is total photosynthate. Estimates were obtained from C isotope balances of shoots, roots and whole plants of sunflower and alfalfa using growth and respiration data collected at intervals of 1 to 2 weeks. e_plant and e_Shoot differed significantly from zero. e_plant ranged between −0.4 and −0.9‰, whereas e_Shoot was much greater (−0.6 to −1.9‰). e_Root was not significantly different from zero. The present results help to resolve the apparent conflict between leaf- and ecosystem-level ¹³C discrimination in respiration.     

Extracellular Matrix in Early Stages of Direct Somatic Embryogenesis in Leaves of Drosera spathulata

The growth and water relations of Paulownia fortunei in photoautotrophic cultures (nutrient medium lacking sucrose and growth regulator) with CO₂ enrichment (PWAH) or without CO₂ enrichment (PWAL) were compared with those in photomixotrophic shoot (PWC; 30 g dm⁻³ sucrose and 0.3 mg dm⁻³ N⁶-benzyladenine) and root cultures (PWR; 0.3 mg dm⁻³ indole-3-butyric acid). The photoautotrophic and photomixotrophic cultures were incubated under photosynthetic photon flux 125 and 60 μmol m⁻² s⁻¹, respectively. 100 % sprouting and significantly higher number of shoots (1.6) were obtained with PWAH as compared to PWAL and PWC. PWAH and PWAL stimulated spontaneous rooting from the cut end of axillary shoots. In PWAH, 84 % of shoots rooted with an average of 5.9 roots per shoot and 4.0 cm of root length in 21 d. Rooting of photomixotrophic shoot cultures were stimulated by an auxin treatment. In this case, 98.3 % of shoots were rooted with an average of 4.6 roots per shoot and 1.9 cm length. A microscopic observation on leaf abaxial surface prints from photomixotrophic shoot and root cultures showed widely open (6 – 8 μm) spherical stomata (12 – 14 μm) and from photoautotrophic cultures elliptical stomata (10 – 12 μm) with narrow openings (3 – 4 μm). Leaves from photomixo-trophic cultures had higher stomatal index as compared to photoautotrophic cultures. The rate of moisture loss from detached leaves was not varying significantly in different cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Micropropagation of Two Australian Native Fruit Species,Davidsonia pruriens and Davidsonia jerseyana G. Harden & J.B. Williams

Nodal explants from in vitro grown seedlings of Davidsonia pruriens and D. jerseyana, established on MS media were treated with various concentrations of three cytokinins. D. pruriens developed optimum shoot growth in terms of shoot height and number of leaves per shoot when 1.0 µM BA was added to basal MS medium while optimum shoot growth for D. jerseyana was obtained when 0.01 µM 2iP was added to the medium. Optimum root initiation and development was obtained when actively growing axillary shoots were cultured on 1/2MS medium plus 32.2 µM IBA for 3–5 days for D. pruriens and 2–3 days for D. jerseyana before transfer to PGR-free medium containing 10 µM riboflavin. Root initiation of more than 80% was achieved with multiple genotypes of D. pruriens and three genotypes of D. jerseyana using juvenile material. The plantlets were transferred to pots and grown in the greenhouse with a success rate of 60% for D. pruriens and 75% for D. jerseyana. Adult D. jerseyana stem explants produced 2–5 shoots per nodal explant upon treatment with 0.1 µM BA. Side shoots from adult D. jerseyana produced similar results for shoot multiplication as for juvenile material. Protocol for multiplication of adult D. pruriens was achieved with much greater difficulty by using material from the green house. Axillary shoots were initiated when 100 µM TDZ was applied to the stem of an adult pot plant and the resultant side shoots were cultured on MS medium containing 1.0 µM BA and 1.0 µM GA₃.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

Sexual reproduction, clonal diversity and genetic differentiation in patchily distributed populations of the temperate forest herb Paris quadrifolia (Trilliaceae)

Clonal plant species have been shown to adopt different strategies to persist in heterogeneous environments by changing relative investments in sexual reproduction and clonal propagation. As a result, clonal diversity and genetic variation may be different along environmental gradients. We examined the regional and local population structure of the clonal rhizomatous forest herb Paris quadrifolia in a complex of forest fragments in Voeren (Belgium). Relationships between population size (the number of shoots), shoot density (the number of shoots per m²) and local growth conditions were investigated for 47 populations. Clonal diversity and genetic variation within and among 19 populations were investigated using amplified fragment length polymorphism markers. To assess the importance of sexual reproduction, seed set, seed weight and germination success were determined in 18 populations. As predicted, local growth conditions largely affected population distribution, size and density of P. quadrifolia. Populations occurring in moist and relatively productive sites contained significantly more shoots. Here, shoots were also much more sparsely distributed compared to populations occurring in dry and relatively unproductive sites, where shoots showed a strongly aggregated distribution pattern. Clonal diversity was relatively high, compared with other clonal species (G/N ratio = 0.43 and Simpson’s D=0.81). Clonal diversity significantly (P<0.01) decreased with increasing shoot density while molecular genetic variation was significantly (P<0.01) affected by population size and local environmental conditions. Lack of recruitment and out-competition of less-adapted genotypes may explain the decreased genetic variation in dry sites. Analysis of molecular variance revealed significant genetic variation among populations (Φ _ST=0.42, P<0.001), whereas pairwise genetic distances were not correlated to geographic distances, suggesting that gene flow among populations is limited. Finally, the number of generative shoots, the number of seeds per fruit and seed weight were significantly and positively related to population size and local growth conditions. We conclude that under stressful conditions populations of clonal forest plant species can slowly evolve into remnant populations characterized by low levels of genetic variation and limited sexual reproduction. Conservation of suitable habitat conditions is therefore a prerequisite for effective long-term conservation of clonal forest plant species.     

Allocation responses to CO2 enrichment and defoliation by a native annual plant Heterotheca subaxillaris

Among plants grown under enriched atmospheric CO₂, root:shoot balance (RSB) theory predicts a proportionately greater allocation of assimilate to roots than among ambient‐grown plants. Conversely, defoliation, which decreases the plant's capacity to assimilate carbon, is predicted to increase allocation to shoot. We tested these RSB predictions, and whether responses to CO₂ enrichment were modified by defoliation, using Heterotheca subaxillaris, an annual plant native to south‐eastern USA. Plants were grown under near‐ambient (400 μmol mol^?1) and enriched (700 μmol mol^?1) levels of atmospheric CO₂. Defoliation consisted of the weekly removal of 25% of each new fully expanded, but not previously defoliated, leaf from either rosette or bolted plants. In addition to dry mass measurements of leaves, stems, and roots, Kjeldahl N, protein, starch and soluble sugars were analysed in these plant components to test the hypothesis that changes in C:N uptake ratio drive shifts in root:shoot ratio. Young, rapidly growing CO₂‐enriched plants conformed to the predictions of RSB, with higher root:shoot ratio than ambient‐grown plants (P < 0.02), whereas older, slower growing plants did not show a CO₂ effect on root:shoot ratio. Defoliation resulted in smaller plants, among which both root and shoot biomass were reduced, irrespective of CO₂ treatment (P < 0.03). However, H. subaxillaris plants were able to compensate for leaf area removal through flexible shoot allocation to more leaves vs. stem (P < 0.01). Increased carbon availability through CO₂ enrichment did not enhance the response to defoliation, apparently because of complete growth compensation for defoliation, even under ambient conditions. CO₂‐enriched plants had higher rates of photosynthesis (P < 0.0001), but this did not translate into increased final biomass accumulation. On the other hand, earlier and more abundant yield of flower biomass was an important consequence of growth under CO₂ enrichment.