• Journal of Korean Society of Forest Science
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• v.78 no.4
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• pp.401-411
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• 1989

The embryos of Pinus taeda, P. rigida, and P. taeda ${\times}$ rigida were cultured for adventitious shoot regeneration in vitro. Culture media were modified from Gresshoff and Doy (MGD), Murashige and Skoog (MMS), Lloyd and McCown (MLM), and Schenk and Hildebrandt (MSH). NAA was added to initiation media at a concentration of 0.1 or 0.01 mg/l. BAP was used at the concentrations of 0.1. 0.5, 1, 2, or 5mg/l. Each explant was induced for 3-4 weeks on solid medium. All explants were cultured up to 16 weeks. Illumination was about $1506{\pm}540lux$ at the level of the tissues in the growth room with a temperature of $25{\pm}2^{\circ}C$. A 16-hour photoperiod per 24 hours was used. Half-strength medium was used for all the subcultures. For shoot production by loblolly pine, MMS, MLM, or MSH is preferred with 5 mg/l BAP with either 0.1 or 0.01 mg/l NAA. For shoot production by pitch pine, MMS, MLM, or MSH is recommended with 2 or 5 mg/l BAP with 0.1 mg/l NAA. For shoot production by the hybrid pine, MMS or MLM is more effective with 1, 2 or 5 mg/l BAP with 0.1 mg/l NAA. There were no differences recognized among the species tried in the patterns of bud formation and shoot development. Different composition of media, in major and minor salts or possibly in vitamins, should be tested for the two developmental stages of adventitious shoots ; the induction of shoot buds and the elongation of them into shoots.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.04a
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• pp.61-62
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• 2003

Clonal propagation of high-value forest trees through somatic embryogenesis (SE) has the potential to rapidly capture the benefits of breeding or genetic engineering programs and to improve raw material uniformity and quality. A major barrier to the commercialization of this technology is the low quality of the resulting embryos. Several factors limit commercialization of SE for Corsican pine, including low initiation rates, low culture survival, culture decline causing low or no embryo production, and inability of somatic embryos to fully mature, resulting in low germination and reduced vigour of somatic seedlings. The objective was to develop a Corsican pine maturation medium that would produce cotyledonary embryos capable of germination. Treatments were arranged in a completely randomized design. Data were analyzed by analysis of variance, and significant differences between treatments determined by multiple range test at P=0.05. Corsican pine (Pinus nigra var. maritima) cultures were initiated on modified !P6 medium. Modifications of the same media were used for culture multiplication and maintenance. Embryogenic cultures were maintained on the same medium semi solidified with 2.5 g/l Gelrite. A maturation medium, capable of promoting the development of Corsican pine somatic embryos that can germinate, is a combination of iP6 modified salts, 2% maltose, 13% polyethylene glycol (PEG), 5 mg!l abscisic acid (ABA), and 2.5 g/l Gelrite. After initiation and once enough tissue developed they were grown in liquid medium. Embryogenic cell suspensions were established by adding 0.951.05 g of 10- to 14-day-old semisolid-grown embryogenic tissue to 9 ml of liquid maintenance media in a 250ml Erlenmeyer flask. Cultures were then incubated in the dark at 2022$^{\circ}$C and rotated at 120 rpm. After 2.53 months on maturation medium, somatic embryos were selected that exhibited normal embryo shape. Ten embryos were placed horizontally on 20 ml of either germination medium ($\frac{2}{1}$strength Murashige and Skoog (1962) salts with 2.5 g/l activated charcoal) or same medium with copper sulphate adjusted to 0.25 mg/1 to compensate for copper adsorption by activated carbon. 2% and 4% maltose was substituted by 7.5% and 13% PEG respectively to improve the yield of the embryos. Substitution of' maltose with PEG was clearly beneficial to embryo development. When 2% of the maltose was replaced with 7.5% PEG, many embryos developed to large bullet-shaped embryos. At latter stages of development most embryos callused and stopped development. A few short, barrel-shaped cotyledonary embryos formed that were covered by callus on the sides and base. When 4% of the maltose was removed and substituted with 13% PEG, the embryos developed further, emerging from the callus and increasing yield slightly. Microscopic examination of the cultures showed differing morphologies, varying from mostly single cells or clumps to well-formed somatic embryos that resembled early zygotic embryos only liquid cultures with organized early-stag. A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also tested. Seedling conversion and growth were highly related to the quality of the germinant at the time of planting. Germinants with larger shoots, longer, straighter hypocotyls and longer roots performed best. When mature zygotic embryos germinate the root emerges, before or coincident with the shoot. In contrast, somatic embryos germinate in reverse sequence, with the cotyledons greening first, then shoot emergence and then, much later, if at all, the appearance of the root. Somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting. Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2002.11a
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• pp.29-30
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• 2002

The goal of this research was to develop the effectiveness of in vitro culture method for A. formosanus and study the environment in vitro conditions affecting on growth. The first series of experiments were examined to investigate the response of three different basal media, MS (Murashige and Skoog, 1962), Knudson (KC; Knudson, 1946) and modified hyponex on growth and multiplication during in vitro culture. Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with BA (1 mg1$\^$-1/) or TDZ (1-2 mg1$\^$-1/). Addition of activated charcoal (1%) to the TDZ containing medium promoted rapid shoot tip proliferation (11.1 shoots per explant) but the same medium had an opposite effect resulting in poor proliferation in the nodal explants. However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 media supplemented with 2% sucrose and 0.5% activated charcoal. Using bioreactor culture for scaling up was also shown the best way for multiple shoot induction and growth of this plant. The second series of experiments was studied to investigate the effect of physical environment factors on growth of in vitro plantlets. The Anoectochilus formosanus plantlets were cultured under different air exchange rate (0.1, 0.9, 1.2h$\^$-1/), without sucrose or supplement 20g.1$\^$-1/ (photoautotrophic or photomixotrophic, respectively), and different photosynthesis photon flux (40, 80, 120 ,${\mu}$mol.m$^2$.s$\^$-1/- PPF). Under non-enrichment CO$_2$ treatment, slow growth was observed in photoautotrophical condition as compared with photomixotrophical condition on shoot height, fresh weigh and dry weight parameters; High air exchange (1.2.h-l) was found to be inadequate for plant growth in photomixotrophical condition. On the contrary, under CO$_2$, enrichment treatment, the plant growth parameters were sharply (visibly) improved on photoautotrophic treatments, especially on the treatment with air exchange rate of 0.9.h-1. The growth of plant in photoautotrophic condition was not inferior compared with photomixotrophic, and the best growth of plantlet was observed in treatment with low air exchange rate (0.9.h-1). Raising the PPF level from 80 to 120${\mu}$mol.m$\^$-2/.s$\^$-1/ decreased the plant height, particularly at 120${\mu}$mol.m$\^$-2/.s$\^$-1/ in photoautotrophic condition, fresh weight and dry weight declined noticeably. At the PPF of 120${\mu}$mol.m$\^$-2/,s$\^$-1/, chlorophyll contents lowed compared to those grown under low PPF but time courses of net photosynthesis rate was decreased noticeably. Light quality mainly affected morphological variables, changes of light quality also positively affected biomass production via changes in leaf area, stem elongation, chlorophyll content. Plant biomass was reduced when A. formosanus were grown under red LEDs in the absence of blue wavelengths compare to plants grown under supplemental blue light or under fluorescent light. Stem elongation was observed under red and blue light in the present experiment. Smaller leaf area has found under blue light than with other lighting treatments. Chlorophyll degradation was more pronounced in red and blue light compared with white light or red plus blue light which consequent affected the photosynthetic capacity of the plant. The third series of experiment were studied to investigate the effect of physical environment factors on growth of ex vitro plants including photosynthesis photon flux (PPF), light quality, growing substrates, electrical conductivity (EC) and humidity conditions. In the present experiments, response of plant on PPF and light quality was similar in vitro plants under photosynthesis photon flux 40${\mu}$mol.m,$\^$-2/.s$\^$-1/ and white light or blue plus red lights were the best growth. Substrates testing results were indicated cocopeat or peat moss were good substrates for A. formosanus growth under the greenhouse conditions. In case of A. formosanus plants, EC is generally maintained in the range 0.7 to 1.5 dS.m-1 was shown best results in growth of this plant. Keeping high humidity over 70% under low radiation enhanced growth rate and mass production.