Li-Chun Huang

Beneficial effects of activated charcoal on plant tissue and organ cultures

SummaryAddition of activated charcoal to the medium for plant tissue cultures improves growth by adsorbing toxic metabolites.

Extended flower longevity of Petunia hybrida plants transformed with boers, a mutated ERS gene of Brassica oleracea

Petunia x hybridaHort.Vilm.-Andr. was transformed with boers, a mutatedallele of BOERS, an ethylene receptor sensor gene ofBrassicaoleracea.boers was obtained by removing anEcoRI cutting site with a silent mutation at Gly-521 andintroducing a point mutation at Ile-62, replacing isoleucine withphenylalanine. Transformation was Agrobacterium tumefaciens mediated.Hygromycin resistant regenerants were tentatively confirmed as transformants byPCRs for HPH and boers and moredefinitively by Southern hybridization of genomic DNA with pBOERS4421. Flowersof transgenic plants retained turgidity and pigmentation considerably longerthan those of untransformed controls, whether left undisturbed on plants orexcised and placed in water. Furthermore, flowers were unaffected by exposureto exogenous ethylene. Excised shoots of transgenic plants released considerablymore ethylene than those of untransformed plants. Transformed plants alsoproduced apparently larger flowers. Unexpectedly higher mortality was observed,suggesting that the ethylene insensitive petunia plants were also lower indisease resistance.

Effects of common components on hardness of culture media prepared with gelrite

SummaryTissue cultures on properly solidified Gelrite media generally showed superior shoot proliferation and rooting, as well as shoot and root vigor and callus development to those on TC agar. Vitrification, or hyperhydricity, was observed in both Gelrite and agar media and minimized by increasing the gel concentrations. Rigidity of Gelrite media depended on combined levels of MS macrosalts, basal nutrient formulations, sucrose concentration, pH, and Gelrite concentration. Most MS macrosalts increased hardness of Gelrite gels; NH4NO3 had a decreasing effect. Rigidity of TC agar gels increased with reductions of MS macrosalts. A slightly softer Gelrite medium resulted when sucrose was excluded. Both Gelrite and agar media were softer at lower pHs and harder at higher pHs. Activated charcoal and mannitol increased gel hardness, and more noticeably of agar gels. NaCl addenda reduced rigidity, with their effects being more pronounced on Gelrite than on agar gels.

High polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture

SummaryTissue browning that frequently results in the early death of bamboo shoots in vitro correlated directly with polyphenol oxidase (PPO, EC 1.10.3.1) activity and inversely with titratable acidity. It was unrelated to the level of endogenous phenols. During the course of culture, timing of PPO activity paralleled that of explant browning. Browning was highest among shoots cultured in a medium of pH 8, which was consistent with the pH optinum of the bamboo enzyme. The pH optimum was first determined with the crude enzyme, then verified with two purified isozymes. Stability of the bamboo PPO was also highest at pH 10. PPO activities of the severely browning Dendrocalamus latiflorus, the moderately browning Phyllostachys nigra, and the relatively non-browning Bambusa oldhamii were inhibited strongly by ascorbic acid, cysteine, sodium diethyldithiocarbamate, and sodium sulfite. But characterization of bamboo PPO according to enzyme inhibitors was not possible because enzyme extracts of the three species gave varied responses to the traditional substances. Nutrient medium addenda of some PPO inhibitors, namely ascorbic acid, cysteine, kojic acid, and thiourea, mainly enhanced browning. However, ferulic acid at 3 mM and lower concentrations reduced the number of brown shoots per culture, although not the percentage of cultures that browned. Polyvinylpyrrolidone failed completely to suppress browning. The two purified isozymes showed different temperature optima for PPO activity: 60°C and 65°C. The purified isozymes displayed a substrate preference for dopamine, or a cathecol oxidase characteristics.

A micropropagation protocol forCinnamomum camphora

SummaryA micropropagation protocol was developed forCinnamomum camphora (L.) Sieb., using as initial explants 3–5-mm shoot tips from newly emerged laterals of 2-yr-old trees. Performance of small shoot tips was compared with that of 2.0-cm nodal segments during subculture. Murashige and Skoog medium (MS) supplemented with different concentrations of N6-benzyladenine (BA) or thidiazuron (TDZ) was used to examine shoot proliferation. In separate experiments, MS was supplemented with 1-naphthaleneacetic acid (NAA) for rooting of shoots, and the commercial preparation EM2 for prevention of hyperhydricity. BA stimulated shoot formation and callus development, whereas TDZ promoted only callus development. Both cytokinins induced hyperhydricity when small shoot tips were used, with severity being directly related to concentrations. Hyperhydricity was avoided in subcultures by using larger nodal segments. EM2 did not alter degree of hyperhydricity but suppressed callus development and strongly promoted shoot multiplication. The number of new shoots after a 6-wk subculture was 9 per nodal segment when supplemented solely with 4.4 μM BA and 18 per segment when further supplemented with 1000 mg EM2 per I. Rooting of shoots occurred best when supplemented solely with 0.54 μM NAA, averaging 7 roots per shoot in 4 wk. Ninety percent of rooted shoots survived transfer to the greenhouse.

Rejuvenation in vitro : modulation of protein phosphorylation in Sequoia sempervirens

Summary Crude extract of whole cells and isolated mitochondria from cultured Sequoia sempervirens shoots of the adult, juvenile, and grafted adult tissues were incubated with [γ- 32 P]-ATP, and the resultant phosphoproteins were then separated by SDS-PAGE and examined through autoradiography. In the extracts of whole cells, the phosphorylation of their 32-kDa protein was evident only in the adult, and that of the 31-kDa protein was detected only in the juvenile cells. Repeated graftings of shoots from adult trees onto juvenile rootstock results in changing the phosphoprotein pattern. It becomes like that of the juvenile tissue and becomes very similar after four grafts. However, no qualitative differences were noted in mitochondrial phosphorylation patterns; rather, the total 32 P incorporation turned out to be lower in the adult tissue than in juvenile and rejuvenated tissues.

Photosynthetic Potentials of in Vitro-grown Juvenile, Adult, and Rejuvenated Sequoia Sempervirens (D. Don) Endl. Shoots

In vitro shoot tips of Sequoia sempervirens (D. Don.) Endl including a juvenile, two adult, and two rejuvenated adult clones were examined for differences in basic physiological characteristics Moisture contents were the same, around 85%, for all tissues regardless of origin Growth rates, determined by fresh weight increase and shoot elongation, were higher for the juvenile and rejuvenated shoots They correlated with higher total nitrogen contents Juvenile and rejuvenated shoots also showed higher rates of photosynthesis and respiration, evidenced by faster O2 evolution and consumption The photosynthetic rates were associated with more chlorophyll, especially chlorophyll a, in the juvenile and the rejuvenated shoots Nevertheless, identical quantum efficiencies of photosystem II indicated the same photosystems were operating and with equal effectiveness in juvenile, adult, and rejuvenated tissues.

Developing an improved in vitro propagation system for slow-growing species using Garcinia mangostana L (Mangosteen)

SummaryThis investigation disclosed that evaluation of tissue culture parameters of slowly developing species (e.g. Garcinia mangostana) requires monitoring of treatments through two or more successive, relatively long passages. Two 8-wk passages were necessary to observe differences in phytohormone effects. Photoperiod and temperature effects were not clearly evident until tissues had been cultured through three passages; the optimal photoperiod and temperature for shoot proliferation could not be established until after the fifth passage. Our investigation revealed that no auxin supplementation was necessary for bud primordium differentiation in cotyledon explants or proliferation of regenerated shoots. The optimum N6-benzyladenine concentration for primordium differentiation was 13.3 μM, and for shoot proliferation ranged from 4.4 to 13.3 μM. Continuous culturing in an 8-h photoperiod at 30°C resulted in progressively intensified degeneration of shoots after three passages. In contrast, successive passages in a 16-h photoperiod/26°C regimen enabled sustained regeneration of shoots. The shoots rooted at a rate of 85% when precultured for 3 d in a medium containing 4921.3 μM indole-3-butyric acid, or 10 d at 492.1 μM, then cultured for two 8-wk passages in phytohormone-free medium. Following acclimatization by gradually lowering the relative humidity in the growth chamber, rooted shoots survived transfer to the greenhouse at a rate of 95%.

A protocol toward multiplication of the medicinal tree, Eucommia ulmoides Oliver

SummaryA protocol based on shoot cultures of 1-mo.-old seedlings was developed for rapid asexual multiplication of eucommia, the source of an antihypertensive medicinal. The explant is an excised shoot tip, 3–5 mm tall. MS basal medium supplemented with 1 mg/liter BA is employed to establish primary cultures and subsequently multiply shoots. Shoots are subculturable on the same medium and can be increased at a rate of 7.5 new shoots per 2-shoot sector every 3 wk. Rooting is achieved in a Gelrite medium with the MS salts reduced to 1/3 strength and the BA replaced by 0.1 mg/liter NAA. The method is not directly applicable to mature trees. Applicability will require explants from rejuvenated sources, possibly attainable by the method of repeated grafting of shoot apices onto juvenile rootstocks, repeated subculturing of shoots, or culturing shoot apical meristems.

Ethylene evolution by juvenile and adult developmental phases of Sequoia sempervirens shoots cultured in vitro

Cultures of juvenile and rejuvenated Sequoia sempervirens shoots generated more ethylene than those of adult shoots. But the higher phytohormone production was only indirectly related to the developmental phase. The juvenile and rejuvenated shoots also grew more rapidly; thus, when measured on a per gram tissue basis the rates of ethylene evolution were the same for tissues of both developmental phases, and even higher for one of the adults. The investigation did not establish whether the faster growth of juvenile and rejuvenated shoots was caused by the ethylene; on the other hand, there was no evidence of inhibitory effects.