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Tree Physiology Advance Access originally published online on December 5, 2008
Tree Physiology 2009 29(1):147-156; doi:10.1093/treephys/tpn013
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Conifer embryogenic tissue initiation: improvements by supplementation of medium with D-xylose and D-chiro-inositol

Gerald S. Pullman1,2, Kelly-Marie Chase3, Anna Skryabina3 and Kylie Bucalo3

1 School of Biology and Institute of Paper Science and Technology, Georgia Institute of Technology, 500 10th Street N.W., Atlanta, GA 30332-0620, USA
2 Corresponding author (jerry.pullman{at}ipst.gatech.edu)
3 Institute of Paper Science and Technology, Georgia Institute of Technology, 500 10th Street N.W., Atlanta, GA 30332, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (LP, Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue and to continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment may decrease this recalcitrance. Sugar and sugar alcohol analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiologic concentrations (Pullman, G.S. and M. Buchanan. 2008. Identification and quantitative analysis of stage-specific carbohydrates in LP (Pinus taeda) zygotic embryo and female gametophyte tissues. Tree Physiol. 28:985–996). Major differences in stage-specific sugars were observed. A simple bioassay was used to evaluate the potential growth promotion of individual carbohydrates added to initiation or multiplication media at physiologic concentrations. Seventeen sugars were screened. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase the initiation of LP. D-xylose and D-chiro-inositol produced statistically significant increases in early-stage embryo growth. When tested for improved initiation in P. taeda, Pseudotsuga menziesii (mirb) Franco and Picea abies L., Karst., D-xylose increased the averages of initiation by 6.5%, 7.3% and 16.7%, respectively. D-chiro-inositol increased the initiation in P. taeda by 7.3% in one test but not in the other, whereas in P. menziesii the initiation increases averaged 8.4% in two tests. Analyses of sugars and sugar alcohols in the seed environment coupled with a bioassay to screen potential media supplements for protocol improvement resulted in statistically significant increases in embryogenic tissue initiation for several coniferous species.

Keywords: embryo development, female gametophyte, loblolly pine, megagametophyte, Pinus taeda, somatic embryogenesis, sugar

Received June 17, 2008; Accepted September 10, 2008


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Forest productivity can be increased by planting tree farms with high-value trees. Methods to propagate large numbers of genetically superior conifer trees are needed. Clonal propagation by somatic embryogenesis (SE) can meet these needs and capture the benefits of breeding or genetic engineering programs to improve wood quantity, quality and uniformity.

Despite the encouraging progress over the past 21 years, since SE was first reported in loblolly pine (LP, Pinus taeda L.), the SE technology has yet to make a significant contribution to the nearly 1.5 billion LP seedlings planted annually in USA (Gupta and Durzan 1987, Schultz 1999). While at least one company has plans to produce several million somatic seedlings in the next few years, the economic feasibility of this propagation system is currently restricted to a fraction of the desired genetic material. Factors currently limiting the commercialization of SE for LP include: (1) low initiation of recalcitrant high-value seed sources, (2) inability to maintain culture growth for many genotypes once initiation has occurred, (3) decline of cultures resulting in loss of plant regeneration potential and (4) low quality of embryos produced resulting in slow initial growth and low germination percentages. Because these factors may be overcome for individual genotypes, they raise the cost of genotypes that can be produced.

The development and improvement of tissue culture protocols is a lengthy and costly process. The traditional literature-based trial and error approach is most often used successfully; however, at some point embryo yield and quality improvements become increasingly difficult to obtain. Additional approaches such as studying natural embryo development to mimic the hormonal (Kapik et al. 1995, Pullman et al. 2003b), nutritional (Carpenter et al. 2000a, 2000b, Pullman et al. 2003c, Silveira et al. 2004, Pullman and Buchanan 2006, 2008, gene expression patterns (Xu et al. 1997, Cairney et al. 2000, 2006, Cairney and Pullman 2007) and physical (Pullman 1997) conditions found in vivo, or understanding how the medium changes over time, such as activated carbon adsorption and pH effects (Van Winkle et al. 2003, Van Winkle and Pullman 2003), can promote protocol development. As the female gametophyte (FG) tissue that normally surrounds and feeds the embryo in vivo is not present during SE in vitro, the addition of compounds that normally are provided by the FG may be necessary for maximal somatic embryo growth.

Sugars and sugar alcohols are the key metabolic components that participate in energy production, provide building blocks for other essential metabolites and help control the cellular osmotic environment (Iraqui and Tremblay 2001). They can be compartmentalized in the vacuole or cytosol or dissolved in the corrosion cavity aqueous environment surrounding the developing embryo (Carman et al. 2005). Sucrose is the most commonly used sugar in vitro, but other mono- and disaccharides including glucose, fructose, maltose and sorbitol are frequently used in plant tissue culture media (Beyl 2000). The choice of carbohydrate often depends on the plant type, explant and tissue age. Myo-inositol, a sugar alcohol, is also included in medium and it is involved in the synthesis of phospholipids, cell wall pectins and membrane systems in cell cytoplasm (Beyl 2000). Pullman and Buchanan (2008) profiled carbohydrates in developing LP embryo and megagametophyte tissue and found large stage-specific differences as the tissue developed. Medium supplementation with approximate physiologic concentrations of individual or combined carbohydrates found in natural seed may improve somatic embryo quality and quantity when used at similar developmental stages. When Pullman et al. (2003b, 2006) had used a similar approach to mimic natural stage-specific abscisic acid or organic acid concentrations in vitro for developing P. taeda somatic embryos, the embryogenic tissue initiation and the early-stage somatic embryo growth were improved. We report here on the beneficial effects of adding D-chiro-inositol and D-xylose to conifer SE initiation and multiplication medium.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Seed carbohydrate analyses to improve embryogenic tissue initiation and embryo development
The following is a tentative step-by-step design of how the authors planned to use carbohydrate analyses information of zygotic embryo and FG tissue to improve the SE protocol.

Phase 1
Analyze FG and zygotic embryos through a sequence of developmental stages to provide stage- and tissue-specific developmental profiles for carbohydrate composition. Repeat the analyses for seeds obtained from two different trees. The results from these analyses are reported in Pullman and Buchanan (2008).

Phase 2
Test individual sugars at approximate physiologic concentrations in an early-stage P. taeda somatic embryo growth bioassay.

Phase 3
If any carbohydrates that cause statistically significant increase in early-stage somatic embryo growth were identified, then test them for the ability to improve embryogenic tissue initiation for immature P. taeda seed.

Phase 4
If P. taeda embryogenic tissue initiation were improved with specific sugar addition, then test the same sugar(s) for ability to improve initiation for other coniferous species.

Growth of P. taeda embryogenic tissue on medium varying in sugar and sugar alcohol content
Early-stage somatic embryos of LP, grown in suspension culture in liquid medium 1133 (Table 1), were used as explants for growth bioassays to evaluate the medium’s potential to support the last phase of initiation and multiplication of embryogenic tissue. The SE cultures were established and grown in liquid medium 1133 (Pullman and Webb 1994, Pullman et al. 2003a). Single stage-2 embryos were isolated with forceps from suspension culture and placed on 2 ml of test growth medium contained in Costar #3526 24-Well Culture Cluster Plates. Explants were grown for 4–7 weeks in the dark at 23–25 °C and then measured for embryogenic tissue diameters. Somatic embryo growth bioassays typically consisted of three genotypes and 40 single early-stage somatic embryos per genotype grown on a test medium arranged in four replicates of 10 embryos each.


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  		Table 1. Media components for initiation experiments.

 
Experiment 1
Embryogenic tissue on initiation or multiplication medium contains somatic embryos homogeneous to zygotic embryos at early-stages 1–3. Individual sugars and sugar alcohols present in FG tissue were calculated for stages 1 and 3 in mM concentrations based on averages for two trees, tissue fresh mass and stage-specific water contents (Table 2). To determine if individual supplemental carbohydrates may have a beneficial effect on early-stage somatic embryo growth, 12 sugars and five sugar alcohols were individually added to medium 1250 (Table 1) at approximate molar concentration found in stage-specific seed tissue. D-chiro-inositol was purchased from Industrial Research Limited (Lower Hutt, New Zealand), glycerol and sucrose were purchased from Mallinckrodt Baker (Paris, KY), and the remaining sugars and sugar alcohols were purchased from Sigma–Aldrich (St. Louis, MO). Due to high cost, D-pinitol, present in stages 1 and 3 at 31.1 and 18.8 mM, respectively, was only tested at 0.5 mM. Tests were not run with fagopyritol B1 because we could not obtain this sugar. If the carbohydrate showed a beneficial response by causing a statistically significant increase in growth, the test was usually repeated. A second trial often repeated and refined the carbohydrate concentrations to determine optimal levels.


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  		Table 2. Calculated early-stage megagametophyte sugar and sugar alcohol mM concentrations and early-stage embryo growth bioassay response to sugars and sugar alcohols.a

 
LP plant materials, media, culture conditions, experimental design and initiation on media supplemented with sugars and sugar alcohols
Cross-pollinated cones were collected in 2006 and 2007 in early- to mid-July from clonal seed orchards, shipped on ice and received within 24–48 h. Cones were stored at 4–5 °C for 1–5 weeks. Cones containing seeds with embryos mostly at stages 2–4 (Pullman and Webb 1994) were used for initiation experiments as described by Pullman et al. (2003d).

Medium 1959 (Table 1) overlaid after 14 days with 0.25 ml of liquid medium 1961 (Table 1) was a starting point for this research (Pullman and Skryabina 2007). Medium pH was adjusted with KOH or HCl after the addition of all ingredients except gelling agent and filter-sterilized materials. Media were autoclaved at 121 °C for 20 min. Aqueous stock solutions of L-glutamine, abscisic acid, brassinolide and {alpha}-ketoglutaric acid (pH adjusted to 5.7) were filter-sterilized and added to the medium that was cooled to about 55 °C. Explants for initiation experiments with zygotic embryos were cultured on 2 ml of medium contained in individual wells of Costar #3526 24-Well Culture Cluster Plates wrapped in two layers of parafilm and incubated at 23–25 °C in the dark. Experiments were composed of 10 replications of 10 explants per test medium and seed source. Treatments were applied in a completely randomized design.

The initiation process for P. taeda is pictured in Pullman et al. (2003d) and described in more detail by Becwar and Pullman (1995). P. taeda initiation occurs in three steps. Extrusion occurs at 1–4 weeks when one or more usually subordinate zygotic embryos expand out of the megagametophyte micropylar end. At 5–7 weeks, proliferating cells and somatic embryos appear in the extruded tissue. During the final step, these multiply to form a mass of embryogenic tissue. These phases can be evaluated as percent of extrusion, percent of explants that form somatic embryos (visible through a dissecting microscope) and percent of cultures achieving a target mass or size. The percent of extrusion and explants with three or more somatic embryos visible under a dissecting scope (i.e., initiation) was routinely evaluated 9–10 weeks after the placement of megagametophytes on the media. Data were evaluated by analysis of variance, and significant differences between the treatment means were determined by the multiple range test at the 5% level of significance using Statgraphics plus Version 4.0. Analyses for extrusion and initiation were done after arcsine {surd}(%) transformation.

Experiment 2
With statistically significant increases seen for arabinose, D-chiro-inositol and D-xylose in the single somatic embryo growth bioassay, the next step was to test the initiation from P. taeda megagametophytes. An embryogenic tissue initiation experiment was conducted with immature seeds from four cross-pollinated seed sources collected in July 2006 and placed onto gelled medium overlaid with liquid medium at 14 days (Pullman and Skryabina 2007). Treatment 1 consisted of control media 1959/1961 (Table 1), treatment 2 (control medium + 375 mg l–1 D-chiro-inositol), treatment 3 (control + 100 mg l–1 D-xylose), treatment 4 (control + 60 mg l–1 arabinose) and treatment 5 (control + 375 mg l–1 D-chiro-inositol + 100 mg l–1 D-xylose). In each case, the indicated sugar alcohol or pentose was added to both the solid and the liquid media.

Experiment 3
Arabinose, D-chiro-inositol and D-xylose were again tested for P. taeda initiation using gelled medium, liquid overlays 1959/1961, and treatments 2–4 indicated in Experiment 2. Four cross-pollinated seed sources collected in July 2007 were used per treatment.

Douglas-fir (DF) plant materials, experimental design and initiation on media supplemented with sugars and sugar alcohols
Experiment 4
With several experiments showing the benefit of addition of D-chiro-inositol or D-xylose, we became interested in the effect of these sugars on other coniferous species. The DF initiation medium of Pullman and Gupta (1994) with agar replaced by 1.8 g l–1 phytagel, addition of 0.1 µM brassinolide, 1.0 mg l–1 abscisic acid, 250 mg l–1 MES, 5.0 mg l–1 biotin, 50 mg l–1 folic acid, 0.48 mg l–1 CuSO4·5H2O, 16.0 mg l–1 ZnSO4·7H2O and 15 g l–1 maltose replacing sucrose was used as treatment 1 (control medium 1789, Table 1) (Pullman et al. 2009). Additional test media included treatment 2 (control + 375 mg l–1 D-chiro-inositol), treatment 3 (control + 100 mg l–1 D-xylose) and treatment 4 (control + 375 mg l–1 D-chiro-inositol + 100 mg l–1 D-xylose). The DF cones containing embryos were collected at stages 3–5 (Pullman and Webb 1994) in early July 2006 from six crosses. The cones were opened and the seeds still attached to the ovuliferous scale were cut from the full ovuliferous scales. The seeds and the attached scale were sterilized as indicated in Pullman et al. (2003d). Under aseptic dissection, the seed coat, integument and nucellus were removed, megagametophytes were carefully cut open and the dominant embryo was lifted out while it was still attached to the FG. The exposed FG and embryo were placed on 7 ml of test medium contained in VWR #29442-036 6-Well Cluster Plates. The plates were wrapped in parafilm and incubated at 23–25 °C in the dark for 7–8 weeks. Six crosses were tested on each medium using six to 12 replicates of six explants per cross.

Experiment 5
Control medium 1789 was further modified to reduce CuSO4·5H2O and ZnSO4·7H2O to 0.024 and 8.0 mg l–1, respectively, and 60.7 mg l–1 pyruvic acid was added (medium 2207, Table 1) (Pullman et al. 2009). Treatment 2 consisted of medium 2207 + 375 mg l–1 D-chiro-inositol, treatment 3 (2207 + 100 mg l–1 D-xylose), and treatment 4 (2207 + 375 mg l–1 D-chiro-inositol + 100 mg l–1 D-xylose). The DF cones containing embryos at stages 3–5 were collected in early July 2007 from six crosses and prepared for culture as indicated above.

Norway spruce materials, experimental design and initiation on medium containing supplemental sugar or sugar alcohol
Experiment 6
Mature Norway spruce (NS) seeds, from F.W. Schumacher Co., Sandwich, MA, were rinsed under cold tap water for 30 min, soaked overnight and sterilized as indicated in Pullman et al. (2003d). Shortly after sterilization, the seeds were dissected, and the integuments and nucellus were removed. The FG was carefully split, the embryo was removed, and the embryo and split gametophyte were placed next to each other on 7 ml modified Verhagen and Wann (1989) initiation medium (medium 1165, Table 1) in 60 x 15-mm Petri plates. Ten replicates of 10 seeds were tested per medium with medium 1165 (treatment 1), treatment 2 (1165 + 375 mg l–1 D-chiro-inositol), treatment 3 (1165 + 100 mg l–1 D-xylose) and treatment 4 (1165 + 60 mg l–1 arabinose). The plates were incubated at 23–25 °C for 16 h of ~ 7 µmol photons m2 s–1 of cool white florescent light for 9–10 weeks. While higher initiation can likely be obtained with other NS protocols, the use of mature seed explants allowed experiments to be conducted at any time during the year.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Growth of P. taeda embryogenic tissue on medium varying in sugar and sugar alcohol content
Experiment 1
Most of the 18 carbohydrates found in FG tissue analyses were tested as supplements to medium 1250 at approximate physiologic concentrations found in early-stage FG tissue (Table 2). Arabinose, D-chiro-inositol and D-xylose showed repeated statistically significant growth improvement when added to the medium. D-xylose showed similar growth increases when autoclaved in medium or added filter-sterilized after autoclaving (data not shown).

LP initiation on medium containing arabinose, D-chiro-inositol or D-xylose
Experiment 2
With early-stage somatic embryo growth increased by arabinose, D-chiro-inositol and D-xylose, we next tested these materials for effect on LP initiation. The percent of extrusion did not differ significantly between treatments (data not shown). However, embryogenic tissue initiation was increased by the addition of 100 mg l–1 D-xylose (treatment 3) and differences were statistically significant (Table 3). D-xylose increased the initiation percentage in three of the four seed sources tested and increased the average initiation from 14.1% (control) to 20.2% (+D-xylose). A greater percentage of extrusions produced embryogenic tissue in the xylose treatment (85%) compared to control treatment at 66%. The addition of arabinose, D-chiro-inositol or a combination of D-xylose and D-chiro-inositol (treatments 2, 4 and 5) did not alter the initiation percentages. It is interesting to note that when D-chiro-inositol and D-xylose were combined, the beneficial effect of D-xylose was lost.


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  		Table 3. Loblolly pine initiation on gelled and liquid overlay media varying in addition of sugar type.

 
Experiment 3
The first four treatments in Experiment 2 were repeated for this experiment. Extrusion was decreased by arabinose (treatment 4) from 50.5% in the control to 43% for the medium containing arabinose and the differences were statistically significant; other treatments did not differ significantly. Both D-xylose (treatment 2) and D-chiro-inositol (treatment 3) caused statistically significant increases in initiation (Table 3). D-xylose raised the initiation from 25.5% in the control medium to 32.8% and increased the initiation in three of the four seed sources tested. D-chiro-inositol increased the initiation to 32.5% with all four seed sources increasing initiation. Conversion of explants with extrusion to explants with somatic embryos increased from 59% in the control treatment to 72% with D-xylose and 66% with D-chiro-inositol.

DF initiation on medium containing arabinose, D-chiro-inositol or D-xylose
Experiment 4
Most explants from one cross were dead and were not included in the statistical analysis. The DF initiation averaged 43.3% across the five living crosses for control (treatment 1) and increased to 55.4% and 55.5%, when D-chiro-inositol (treatment 2) or D-xylose (treatment 3) was added (Table 4). D-chiro-inositol increased initiation for all five cross-pollinated seed sources tested and the differences were statistically significant. D-xylose increased the initiation for four of the five crosses tested and again differences were statistically significant. Average embryogenic tissue masses transferred were also increased about 17% by D-chiro-inositol and 13% by D-xylose; the differences were statistically significant at P = 0.05 for D-chiro-inositol but not for D-xylose (Table 4).


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  		Table 4. Douglas-fir initiation on medium varying in sugar type.

 
Experiment 5
The addition of 100 mg l–1 D-xylose (treatment 3) again increased the initiation in five of the six crosses tested and increased the average initiation from 37.3% in the control treatments to 44.5% in the xylose treatments (Table 4). The addition of 375 mg l–1 D-chiro-inositol (treatment 2) also resulted in statistically significant increases in the initiation to 45.6% with all crosses responding favorably (Table 4). As was seen in the LP experiments, the combination of D-chiro-inositol and D-xylose canceled the beneficial effect of either carbohydrate.

NS initiation on medium containing arabinose, D-chiro-inositol or D-xylose
Experiment 6
Embryogenic tissue for Picea abies L., Karst. began to form after about 4 weeks. Evaluations after 9–10 weeks showed increased initiation on medium containing D-xylose (treatment 3). D-xylose more than doubled the initiation, and differences were statistically significant (Table 5). Media containing arabinose (treatment 2) or D-chiro-inositol (treatment 4) did not improve the initiation.


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  		Table 5. Norway spruce initiation on medium varying in sugar type using mature zygotic embryos.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The LP cells can grow on different carbohydrate substrates. Vuke and Mott (1987) had found that 13 carbohydrate substrates, namely ribose, glucose, fructose, galactose, mannose, maltose, trehalose, melibiose, lactose, cellobiose, gentibiose, raffinose and melizetose, had supported the growth over two 4-week subcultures in LP callus that was at least 75% of the growth that occurred on sucrose alone.

To guide medium development using analytic data, authors used a control medium for early-stage somatic embryo multiplication that contained 92 mM sucrose and 5.5 mM myo-inositol (medium 1250, Table 1). This decision was based on the knowledge that the medium worked well to multiply somatic embryos of LP and that sucrose was found in early-stage zygotic embryos at 14–27.2 mM along with myo-inositol present at 0.15–0.06 mM. Individual carbohydrates would be added to this medium at approximate concentrations found in FG at stage 1 or 3. As most sugars were found in zygotic tissue at low mM concentrations, adding them to the control medium would cause minimal change in medium osmotic potential. An alternative approach would be to test individual sugars as the sole carbohydrate. This would cause significant change in medium osmotic potential, and data from Vuke and Mott (1987) indicated that sugars such as arabinose or xylose were toxic to the pine tissue when they are used as a sole carbohydrate source. Because the FG provides nutritional and regulatory materials to the developing embryo, a decision was made to use analytic values from FG tissue as the guide for bioassay tests. Carman et al. (2005) suggest that analyses of corrosion cavity fluids surrounding the embryo provide a more accurate picture of embryo nutrition.

Early work by Strickland et al. (1987) on the benefit of maltose in alfalfa SE stimulated other researchers to test maltose for improved tissue culture protocols. Since then many researchers had found maltose to be useful to improve many tissue culture steps, particularly during SE. Maltose is often the sugar of choice for the initiation of embryogenic tissue for many coniferous species (Nagmani et al. 1993, Gupta et al. 2004, Salajova and Salaj 2005, Steiner et al. 2005). The LP embryogenic tissue formation occurs on sucrose (Becwar et al. 1990), but increased initiation has been shown to occur on maltose and galactose (Pullman and Johnson 2002, Denchev et al. 2004). Maltose was present in early-stage FG 1 and 3 at 0.02 and 0.07 mM, respectively, and probably resulted from breakdown of starch. It is interesting to note that maltose was not stimulatory in our bioassay approach when tested at physiologic concentrations present in early-stage zygotic embryos.

D-xylose caused stimulation of early-stage somatic embryo growth in bioassays (added to sucrose-containing medium) and increased embryogenic tissue initiation in P. taeda (maltose-containing medium), Pseudotsuga menziesii (mirb) Franco (maltose-containing medium) and P. abies (sucrose-containing medium). D-xylose was most stimulatory when supplemented to medium at 0.67 mM compared to the presence in LP FG from stages 1 and 3 at 0.20 and 0.09 mM, respectively. D-xylose, commonly known as wood sugar, is found in plant tissues, and the polymers of xylose may comprise 25% of tissue dry mass (Zablackis et al. 1995). While xyloglucans are involved in load-bearing due to their ability to cross-link cellulose microfibrils, xylose may also have a role in embryogenesis. Mollard et al. (1997) isolated xylose-rich polysaccharides from primary walls of embryogenic cell lines of Caribbean pine. Malinowski and Filipecki (2002) and Malinowski et al. (2004) had found that xyloglucan endotransglucosylases/hydrolases (XTHs) act during induction of SE in the early stages of embryo development in cucumber cell suspension. Several XTHs were differentially expressed after the induction of cucumber SE. The XTHs cleave xyloglucan chains and have the ability to insert them at a different site and may also produce oligosaccharide chains that participate in the transduction of intercellular signals (Fry et al. 1993, Creelman and Mullet 1997). LP medium is often used in SE research on P. abies and other coniferous species and contains 0.5 mM L-arabinose and D-xylose (Von Arnold and Eriksson 1981).

Two percent myo-inositol (111 mM) was originally included in LP initiation medium to raise osmolality from about 120–230 mmol kg–1 based on measured water potential of developing P. taeda megagametophytes (Pullman 1997, Pullman and Johnson 2002). The LP analyses showed 0.15 and 0.06 mM myo-inositol in stages 1 and 3 FG, respectively. Carman et al. (2005) had performed the analyses of DF corrosion cavity fluids and found that the levels of cyclitols, sucrose equivalents, erythrose and arabinose were manyfold higher in corrosion cavity fluid than in the whole-seed tissues. Combined sugars analyzed (excluding unidentified sugar alcohols and longer chain oligosaccharides) accounted for 12.8% of the corrosion cavity sample dry mass, myo-inositol accounted for 7.2% and sucrose equivalents accounted for 4.1%. Assuming 85% water content for corrosion cavity fluid, millimolar calculations were about 60 mmol l–1 for myo-inositol, 18 mmol l–1 for sucrose equivalents and 12 mmol l–1 for other sugars (Carman et al. 2005).

The LP FG has two peaks in D-chiro-inositol content: one during early and another in mid- to late-development (Pullman and Buchanan 2008). D-chiro-inositol present in the early-stage FG may support early-stage embryo development or provide a substrate for the synthesis of fagopyritols that are present in the early-stage FG (Ma et al. 2005). In soybean, there is no evidence for the synthesis of D-pinitol and D-chiro-inositol in somatic embryos (Gnomes et al. 2005). Feeding studies show that myo-inositol, D-chiro-inositol and D-pinitol are synthesized in maternal tissues and are directly unloaded from seed coats to soybean embryos. Fagopyritols and galactopinitols are synthesized in embryonic tissues from transported D-chiro-inositol and D-pinitol, and the accumulation of these compounds in the maturing seed is limited by the supply of D-chiro-inositol and D-pinitol to the embryo (Gnomes et al. 2005). Gosslova et al. (2001) had found the presence of D-pinitol in the developing whole seeds and in mid-stage to full-term FG and embryos of NS but when Lipavaka et al. (2000) measured the sugars in NS somatic embryos, D-pinitol was not present in any stage examined. However, somatic embryos of P. taeda were found to contain different levels of myo-inositol, D-chiro-inositol and fagopyritol B1, depending on the maturation treatment (Carpenter et al. 2000b). Many tests combining D-chiro-inositol and D-xylose or arabinose, D-chiro-inositol and D-xylose did not further improve initiation and often reduced the benefit seen with D-chiro-inositol, D-xylose or arabinose alone (Tables 3 and 4). Due to lower cost, we preferred to use D-xylose for initiation in both LP and DF.

Although arabinose showed increased growth in several LP early-stage somatic embryo growth bioassays, it did not improve the initiation for either LP or NS. The work of Carman et al. (2005) shows high levels of arabinose present in the seed corrosion cavity during embryo development, whereas we did not test the effect of arabinose in DF due to the negative results with LP and NS. Arabinogalactan proteins, chitinases and lipochitooligosaccharides have been found to stimulate embryogenesis in angiosperms and gymnosperms (Cairney and Pullman 2007).

Media improvements most often occur from empirical modifications of the existing basic formulations. This approach is inefficient, costly, and does not always lead to the desired improvement. Several media for coniferous plants were developed with the aid of tissue analyses. Litvay et al. (1985), Teasdale et al. (1986) and Pullman et al. (2003c) had used conifer seed metal analysis to formulate new culture media for growing embryogenic and non-embryogenic cultures of LP and radiata pine. Pullman et al. (2006) used organic acid analyses to improve the embryogenic tissue initiation in LP. Here, we formulated several new embryogenic tissue initiation media for various coniferous species based partly on the analyses of P. taeda seed tissues for carbohydrate content. The data presented in this paper continue to support the concept that fundamental analyses of seed and embryo tissues during the course of development can lead to tissue culture protocol improvements.


   Acknowledgements
 
The authors thank the member companies of the Institute of Paper Science and Technology at Georgia Tech and the State of Georgia TIP3 Program for financial support, and Weyerhaeuser Company for cone collections. In addition, they are grateful for the valuable technical assistance of J. Dreger, E. Ford, K. Golson, J. Grabowski, Shannon Johnson, S. Lukjan, K. Olsen and C. Umejiego.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

    Becwar M.R., Pullman G.S. Somatic embryogenesis in loblolly pine (Pinus taeda L.). In: Somatic Embryogenesis in Woody Plants – Gymnosperms—Jain J.S., Gupta P.K., Newton R.J., eds. (1995) Vol. 3. The Netherlands: Kluwer. 287–301.

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G. S. Pullman and S. Johnson
Osmotic measurements in whole megagametophytes and embryos of loblolly pine (Pinus taeda) during seed development
Tree Physiol, June 1, 2009; 29(6): 819 - 827.
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 Tree PhysiolHome page
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Loblolly pine (Pinus taeda) female gametophyte and embryo pH changes during seed development
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