Patent Abstract:
according to the invention an embryogenic cell mass is cultured with a culture medium comprising an anti - auxin resulting in an unexpected shift in physiology from proliferation to maturation . proliferation is reduced so that the formation of new immature embryos ceases . reduction of proliferation facilitates the transition from proliferation to maturation and maturation frequency is increased to a much larger extend than expected . surprisingly , it has been discovered that the quality of the somatic embryos is not reduced , although the activity of the important endogenous plant growth regulator , auxin , is reduced . as a mater of fact , the overall quality of the mature embryos harvested at the end of maturation is actually increased over the prior art .

Detailed Description:
the invention is now described in more details using a number of examples and the following figures : fig1 . photograph of rapidly growing abies nordmanniana cell line disclosing maturation according to the invention and according to prior methods . fig2 . photograph of slowly growing abies nordmanniana cell line disclosing maturation according to the invention and according to prior methods . fig3 . graphic illustration of preferred embodiments of the anti - auxin phase according to the invention . fig4 preferred and non - preferred embodiments of an embryo according to the invention . fig5 . photograph of abies nordmanniana cell line matured according to different embodiments of the invention . fig6 photograph of abies nordmanniana cell line matured according to different embodiments of the invention . fig7 . graphic representation of the endogenous indole acetic acid ( iaa ) level in three cell lines of abies nordmanniana . fig8 . photograph of germination and hardening of embryos according to the invention . fig9 . photograph of three mature somatic embryos of abies nordmanniana ; left picture showing typical embryo from treatment b ), center picture showing typical embryo from treatment d ), and right picture showing typical embryo from treatment c ). the inventive idea has been tested in a number of different situations , with different conifers and cell lines of various characteristics , such as newly established cell lines and older cell lines , slowly growing cell lines , rapidly proliferating cell lines , transgenic cell lines , cell lines that can produce mature embryos according to known methods ( such as a - type cell lines ), and cell lines that do not produce mature embryos according to known methods ( such as b - type cell lines ). three different cell lines of abies nordmanniana were used in the present study . the tested cell lines have been in continuous culture for several years . the cell line g . 3 . 8 was initiated from an immature zygotic embryo in 1989 on half strength ms medium ( murashige and skoog , 1962 ) as described previously ( nørgaard and krogstrup , 1991 ), cell line g . 35 . 8 was initiated from a mature zygotic embryo on sh medium in 1991 ( nørgaard and krogstrup , 1995 ), and cell line g . 78 . 14 was initiated in 1993 from an immature zygotic embryo on modified half strength blg medium ( verhagen and wann , 1989 ). from 1996 , all cultures were maintained by biweekly subculture onto fresh half strength modified blg medium and grown at 24 +/− 1 ° c . in the dark . cell lines g . 3 . 8 and g . 78 . 14 proliferated fast on maintenance medium with a doubling time of the weight shorter than 2 weeks . cell line g . 35 . 8 proliferated relatively slowly with a doubling time of the weight higher than 2 weeks . the half strength ms medium ( murashige and skoog , 1962 ) contained half strength ms macro , micro and feedta . the ms vitamins were modified by 10 × thiamine - hcl , 100 mg / l inositol , 500 mg / l l - glutamine and casein hydolysate . the sh medium ( schenck and hildebrandt , 1972 ) was modified by inclusion of 500 mg / l l - glutamin and casein hydrolysate . the half strength blg medium ( verhagen and wann , 1989 ) was modified from half strength ms medium by omitting nh 4 no 3 and reducing kno 3 to 50 mg / l . kcl was added at 372 . 5 mg / l , l - glutamine at 750 mg / l and l - aspargine at 50 mg / l . vitamins were modified by use of 10 × thiamine - hcl , 100 mg / l inositol . for all media 1 . 8 g / l phytagel ( gellan gum ) was used as gelling agent and ph was adjusted to 5 . 7 prior to autoclaving . for proliferation , sucrose was used in a concentration of 1 % and bap in a concentration of 5 μm . no auxin was used in the maintenance medium . the amino acids were prepared as a filter sterilised stock solution and added after autoclaving and cooling of the medium to about 50 ° c . the standard maturation medium ( the control ) is modified from the half strength blg proliferation medium mentioned above . the medium does not include sucrose and bap , but is supplemented with 45 g / l maltose ( merck 5911 ), 50 g / l peg - 4000 ( fluka ), and 40 μm aba ( sigma a1049 ). the choice of these three culture medium components is determined primarily by the conifer species in question . for abies nordmanniana it is known that superior maturation is achieved by using maltose instead of sucrose as a carbohydrate source . similarly it is known that addition of peg - 4000 improves maturation . still it may be possible to obtain just as good results with other combinations of these components and also by using other components with equivalent effects . similarly , the half - strength blg medium has been chosen , because it has been found suitable for many conifers including abies spp , picea spp and pinus spp . an equivalent basal medium may be used to obtain results that are equivalent to the results obtained with blg medium , the important issue being that the basal medium should be balanced to meet the requirements of the conifer species in question . in this experiment , the standard maturation medium was modified by excluding peg and by including one of the following 3 chemicals , that are traditionally expected to affect the endogenous concentration of the naturally occurring auxin iaa or to affect the normal effect of auxin in plant cells : 1 ) pcib ( auxin - antagonist ) in a concentration of 25 or 117 μm , 2 ) tiba ( auxin transport inhibitor ) in a concentration of 5 or 50 μm , 3 ) phlouroglucinol : ( inhibitor of auxin degradation ) ( see george 1993 , page 429 bottom - 430 ) in a concentration of 30 mm . in addition to these modifications the standard maturation medium was modified by : 4 ) reducing the concentration of boron ( precursor of indole acetic acid synthesis ) to 10 μm , i . e . 20 % of the normal concentration and finally by 5 ) including 25 μm pcib in medium with no aba . the cultures were matured in the dark at 24 ° c .+/− 1 ° c . the anti - auxins and means to reduce endogenous auxin effects have been chosen to represent different groups of treatments or compounds . thus the experiment represents very different ways of reducing auxin concentration or reducing auxin effects as well as the necessary controls and a treatment that is expected to increase endogenous auxin concentration . two weeks after the last subculture , 4 grams of embryogenic cells was transferred to a 250 ml flask ( blue cap ) together with a magnet stirrer . 100 ml of liquid proliferation medium was added , and the suspension was stirred for 5 minutes at high speed on a magnet stirrer to obtain a homogenous suspension of cells and cell clumps . the culture was then pre - cultured on a gyrotory shaker ( 120 rpm ) for one week at 24 +/− 1 ° c . in the dark . following the pre - culture , the culture was stirred again on a magnet stirrer at high speed for 5 minutes . the culture was transferred to a 100 ml measuring cylinder and allowed to settle for 30 min . the supernatant was discharged and from the remaining culture aliquots of 1 ml were transferred with a cut pipette tip onto filter paper ( whatmann # 2 ) placed on 25 ml of maturation medium in 10 cm petri dishes ( nunc , denmark ). the filters containing the maturing somatic cultures were transferred onto fresh medium every 2 weeks . the purpose of plating the cultures onto the maturation medium is to get an uniform spread of the plated cells on the solid support ( in this case filter paper ) in order to increase reproducibility and reduce variation . maturation may also simply be initiated by transferring clumps of embryogenic cell mass to the maturation medium . well - developed mature embryos were collected for cold treatment after about 20 weeks of maturation . the isolated mature embryos were transferred to a nylon filter that was placed on a foam rubber pad in a high petri dish . a wetted filter paper was placed in the bottom and in the lid of the petri dish . the petri dish was closed , sealed with two rounds of polyethylene film , wrapped with aluminium foil , and then placed at 5 ° c . in the dark for 4 weeks for cold treatment . after cold treatment , the nylon filter with the mature embryos was transferred to bgm - 2 medium ( krogstrup et al , 1988 ). the bgm - 2 medium included 20 g / l sucrose , 10 g / l activated charcoal ( merck 5411 ), and was galled with 3 . 5 g / l phytagel ( gellan gum ). the petri dishes were placed in a growth room at 20 ° c . with a 16 hour photoperiod . after one week of germination , the nylon net was removed and the embryos were placed in direct contact with the medium . after two weeks of germination , embryos with a root were placed vertically with the root submerged in the medium . later , the germinating embryos were transferred to soil and placed in a controlled greenhouse . the described method for germination is just one of many different methods that can be used to germinate conifer somatic embryos . thus , it may also be found advantageous to include a desiccation step between the embryo maturation and germination . during this step the embryo may be subjected to controlled desiccation in order to simulate the maturation drying taking place during the corresponding last phase of seed maturation . the water content of mature somatic embryos and embryos from seeds was measured by weighing approximately 25 embryos ( fresh weight , fw ), drying the embryos at 60 ° c . for two days and weighing the embryos again ( dry weight , dw ). the water content is the calculated on a fresh weight basis : ( fw − dw )/ fw . fig1 and 2 disclose maturation of two embryogenic cell lines of abies nordmanniana . cell line g . 3 . 8 ( fig1 ) is one of the cultures that proliferate rapidly on proliferation medium . according to prior methods it produces no or very few somatic embryos . cell line g . 35 . 8 ( fig2 ) is one of the cultures that proliferate relatively slowly on proliferation medium . it does produce a few mature somatic embryos according to prior methods . four different protocols for maturation were tested : a ) maturation for 20 weeks on maturation medium including 40 μm aba and 5 % peg - 4000 ( control method ). b ) maturation as for a ), but from week 4 to 8 the cultures were matured on medium including 40 μm aba and 25 μm pcib . c ) maturation for 20 weeks on maturation medium including 40 μm aba and 25 μm pcib . d ) maturation for 20 weeks on medium including no aba but 25 μm pcib . fig3 shows the number of well developed mature somatic embryos per petri - dish in cell lines g . 3 . 8 and g . 35 . 8 after 20 weeks of maturation according to the four different maturation protocols describes above . for the tested embryogenic cell lines of abies nordmanniana , only few mature embryos were formed on the control medium with aba and peg ( fig1 a , 2 a and 3 ). in most cases , maturation of embryos was initiated in these cultures , but due to proliferation of the surrounding tissue the developing embryos were overgrown and they did not develop further . on medium including both aba and 25 μm pcib , proliferation was reduced and high numbers of embryos developed during maturation ( fig1 b 2 b and 3 ). however , the number of well - developed mature embryos was dependent on both the concentration and of the application period of pcib ( fig3 ). some of the tested cell lines proliferated very fast whereas other cell lines only proliferated slowly on the maintenance medium . these characteristics were also found when the cell lines were grown on maturation medium , and the rate of proliferation during maturation was strongly cell line dependent . the optimum application of pcib during maturation was found to be determined partly by the rate of proliferation of the cell line , and therefore a general protocol for the application of pcib covering all tested cell lines could not be found . for illustration , results are shown for a ‘ fast ’ and for a ‘ slow ’ proliferating cell line . cell line g . 3 . 8 proliferated fast on maintenance medium and on maturation medium including aba and peg and no well formed mature embryos developed during maturation on medium without pcib ( fig1 a and 3 ). when 25 μm pcib was included into the medium from week 4 to 8 of the 20 week maturation period , about 40 well formed embryos developed and proliferation was reduced slightly ( fig1 b and 3 ). however , through inclusion of 25 μm pcib during the entire maturation period about 125 well - formed embryos developed per petri dish and proliferation was strongly reduced ( fig1 c and fig3 ). cell line g . 35 . 8 proliferated slowly on maintenance medium and also during maturation on medium including aba and peg . only few mature embryos developed on the control medium ( fig2 a and 3 ), and inclusion of pcib into the maturation medium had a clear positive effect on the number of well formed embryos for this cell line . however , best results were found when pcib was only included into the maturation medium from week 4 to 8 of the 20 week maturation period where about 250 embryos developed per petri dish ( fig2 b and 3 ). opposite to cell line g . 3 . 8 , inclusion of pcib during the entire maturation period had a negative effect on the number of well formed mature embryos of cell line g . 35 . 8 ( fig2 c and 3 ) and only about 15 well formed embryos developed per petri dish . if aba was excluded from maturation medium including 25 μm pcib , a relatively high number of embryos started to mature in the cultures , but only few and clearly abnormal and precociously germinating embryos were developed on this medium for all tested cell lines ( fig1 d and 2 d ). since practically no maturation takes place on medium without both anti - auxin and aba this clearly indicates that anti - auxins in themselves play a role in embryo maturation . as in the other cases of inclusion of pcib , proliferation was reduced . fig4 discloses the morphology of mature somatic embryos of abies nordmanniana and typical abnormalities caused by over - exposure to pcib during maturation . a : well developed embryo with a straight axis from root to shoot and approximately 5 cotyledons surrounding the shoot meristem . b : embryo with only two cotyledons . the embryo is relatively symmetric around the vertical axis , and the shoot meristem is normally intact . c : embryo with two cotyledons . the embryo is clearly assymetric with a big and a small cotyledon . the shoot meristem is affected by the abnormality . d : embryo with only one cotyledon . the embryo has no functional shoot meristem . the well developed mature somatic embryo of abies nordmanniana , has a straight axis from root to shoot pole , and about 5 cotyledons ( fig4 a ). when cell lines were grown on maturation medium including pcib in supra optimum concentrations or for some cell lines in too long periods , high numbers of abnormal mature embryos developed . a very characteristic abnormality was a reduction in the number of cotyledons ( fig4 b - d ). when the slowly proliferating cell lines were grown on medium containing aba and 25 μm pcib for longer periods than 4 weeks , embryos with only two symmetric cotyledons developed ( fig4 b ). when higher concentrations than 25 μm of pcib was used for the tested cell lines , the embryos developed asymmetrically with one major and one minor cotyledon ( fig4 c ) or with only one cotyledon ( fig4 d ). the shoot meristem seemed to be intact in mature embryos with two symmetric cotyledons and these embryos could germinate and develop further , but mature embryos with two asymmetric or only one cotyledon did not germinate properly . fig5 discloses maturation of cell line g . 78 . 14 . this cell line proliferates rapidly and produces very few normal mature embryos according to prior methods . the cell line was matured for 20 weeks on medium comprising 40 μm aba and a ) 25 μm pcib , b ) 0 . 4 mm phloroglucinol , or c ) reduced concentration of boron ( 20 % of normal concentration ). for control see fig1 a or 2 a . medium with a decreased concentration of boron also had a positive effect on the number of mature embryos ( fig5 ). on maturation medium with a decreased concentration of boron , a clear increase in the number of developing mature embryos was seen in some of the tested cell lines . however , proliferation continued in these cultures and overgrowth of the developing embryos reduced the number of well - developed embryos . as reduced boron was only tested at one concentration it is possible that positive effects could be obtained by optimising the concentration of boron . when embryo maturation was performed on medium including phloroglucinol ( 0 . 4 mm ) no well developed embryos were found and proliferation was increased in the tested cell lines ( fig5 ). phloroglucinol is supposed to act as an inhibitor of auxin degradation and the addition of phloroglucinol is thus supposed to cause an increase in endogenous auxin concentration . fig6 discloses maturation of cell line g . 78 . 14 for 20 weeks on maturation medium comprising 40 μm aba and a ) 2 . 5 mg / l tiba ( 5 μm ), or b ) 25 mg / l tiba ( 50 μm ). for both of the tested concentrations of the auxin - transport inhibitor , tiba , a strong reduction of the proliferation on the maturation medium was found for all tested cell lines , but only very few , and mostly abnormal , mature embryos developed ( fig6 ). since tiba was only tested in two concentrations ( 5 and 50 μm ) one cannot exclude the possibility that a positive effect could be obtained with a lower concentration . fig7 discloses the endogenous concentration of indole acetic acid in three cell lines of abies nordmanniana . the concentrations were measured during proliferation of the cultures . the bars represent the standard error . endogenous iaa was found in proliferating cultures of all 3 tested cell lines ( fig7 ). the concentration of iaa was found to be in the range from 6 to 9 nmol / mg dry weight in the cultures but no significant difference was found between the ‘ fast proliferating ’ and the slower proliferating cell line . the water content of mature zygotic embryos of abies nordmanniana was about 30 %. the water content of mature somatic embryos was genotype dependent , but for both cell lines the water content of somatic embryos were higher than the water content of zygotic embryos . for somatic embryos from maturation medium with pcib , the water content was between 65 and 75 %, which is lower than in the corresponding mature somatic embryos from maturation medium without pcib . fig8 a discloses germinating embryos produced according to the invention . the embryos have been subjected to cold treatment for two weeks followed by two weeks of germination on germination medium . fig8 b discloses germinated plants according to the invention transferred to soil for hardening . high numbers of mature embryos was developed from several cell lines with the use of pcib in the maturation medium . germination of embryos was fine and close to 100 % of the embryos developed both shoot and root , and more than 2000 of the plants were transferred to soil and into the greenhouse ( fig8 b ). regenerated plants were generally dormant as is the case with seedlings of the same species . the plants survived in the soil , but it was very difficult to obtain further growth from the bud . the problem was probably related to dormancy and lately it has been possible to induce further growth by optimisation of the germination protocol . it is known that subjecting such dormant plants to conditions of short days and low temperatures for a period of time to be determined experimentally induces bud flushing and thereby continued growth . approximately 50 cell lines were initiated from mature seeds in the same way as cell line g . 78 . 14 ( example 1 ). the cultures were initiated and maintained on modified half strength blg medium ( see example 1 ). the cultures were less than 6 months old when they were subjected to maturation . the cultures were plated as described in example 1 . the cultures were subjected to two different maturation protocols . 1 ) maturation for 8 weeks on culture medium comprising 40 μm aba and 5 % peg - 4000 . 2 ) maturation for as 1 ) but from week 2 to 4 the cultures were on culture medium comprising 40 μm aba and 25 μm pcib . preliminary experiments had shown that it was detrimental to subject these newly established cell lines to continuous anti - auxin . therefore only one treatment comprising a short anti - auxin step was included . first of all it should be noted that the newly established cell lines require substantially shorter time for maturation than the older cell lines . part of the cell lines produced high numbers of well developed mature somatic embryos according to the control treatment ( treatment 1 ). the remaining cell lines did not . when subjected to the two weeks anti - auxin step described above , almost all cell lines produced high numbers of mature somatic embryos according to the invention . thus the outcome was that it was possible to produce mature somatic embryos from all tested cell lines . three embryogenic cell lines , d . 2 . 9 , d . 4 . 1 , d . 7 . 1 , of picea abies , initiated from immature seeds in 1989 and kept in cryostorage until 1998 as described by nørgaard et al ( 1993 ) were used for the present experiment . the cultures were maintained on semi - solid proliferation medium bmi - s1 ( krogstrup 1986 ) gelled with 1 . 8 g / l gelrite by biweekly sub - cultures . prior to transfer to maturation medium , 4 grams of cell mass from each cell line was weighed off and transferred to 50 ml liquid proliferation medium in a 250 ml erlenmeyer flask . the flask also contained a sterile magnet . to disintegrate the cultures they were stirred for 5 minutes using a magnet stirrer . then the flasks were shaken for 15 minutes on a rotary shaker at 175 rpm . the medium with suspended cell aggregates were subsequently poured into a 100 ml measuring cylinder and allowed to settle for 30 minutes . the supernatant was discarded and aliquots of 1 ml of cells were plated on sterile whatman # 54 paper discs placed an the various maturation media . three replicate dishes were made for each combination of cell line and treatment . a : maturation on bmg - 1 medium ( krogstrup et al 1988 ) modified by increasing the aba concentration to 40 μm . b : as a , but from week 2 to week 4 maturation on culture medium comprising 2 . 5 μm pcib and 40 μm aba . c : as a , but from week 2 to week 4 maturation on culture medium comprising 5 . 0 μm pcib and 40 μm aba . d : maturation on medium comprising 2 . 5 μm pcib and 40 μm aba . e : maturation an medium comprising 5 . 0 μm pcib and 40 μm aba . cell lines d . 4 . 1 and d . 7 . 1 which are typical so - called a - type cell lines produced mature somatic embryos without any anti - auxin step . however , they produced higher numbers of somatic embryos when an anti - auxin step was included . the effect of the anti - auxin step was to reduce the proliferation during maturation . cell line d . 2 . 9 does not produce as many embryos as the other two cell lines . when matured without an anti - auxin step , the culture gradually turns brown and looses viability . mature embryos are then produced . when subjected to an anti - auxin step the culture also initially turns brown , but later new clumps of fine , translucent proliferating embryogenic cell mass is produced at the periphery of the culture . from this cell mass , a high number of mature somatic embryos are in turn produced . thus it seems that the inclusion of an anti - auxin step has the effect of changing cell lines that are not capable of producing mature somatic embryos into cell lines that can do this . clumps of the newly formed cell masses from d2 . 9 have been transferred back to proliferation medium and in this way it has become possible to produce mature embryos from this culture again . it is therefore possible to use one aspect of the method according to the invention to improve the ability of cell lines to produce mature somatic embryos . this can evidently be done by subjecting the cultures to the conditions described above for cell line d . 2 . 9 , but it can likewise be done by incorporating an anti - auxin step in the proliferation step or in a step between proliferation or maturation . two cell lines of picea sitchensis , g . 1 . 12 , g . 1 . 13 , initiated from cotyledons from mature embryos were used for the present study . the cultures were initiated and pretreated on bmi - s1 medium as above , but with 1000 mg / l casein hydrolysate and 10 μm 2 , 4 - d . pretreatment , maturation medium and anti - auxin step as for picea abies . both the tested cell lines produced high numbers of mature somatic embryos both with and without an anti - auxin step . however , proliferation was clearly reduced by subjecting the cultures to an anti - auxin step , and the general appearance of the cultures was improved . transformation of embryogenic abies nordmanniana cell cultures and subsequent regeneration of plants embryogenic cell lines of abies nordmanniana were transformed biolistically using the method disclosed by walter et al ( 1998 ) with a few modifications . firstly , the species is abies nordmanniana in stead of pinus radiate . the modified half strength blg medium described in example 1 above was used for proliferation and also during the selection step after transformation . the plasmid transferred was the pcw 122 plasmid containing the reporter gene uida under the control of the camv 35s promoter and the npt ii selectable marker gene controlled by the camv 35s promoter . selection was carried out on proliferation medium comprising 50 mg / l geneticin . expression of the uida reporter gene was detected histochemically in transformed embryogenic tissue , in derived mature somatic embryos produced according to the present invention and in regenerated plants likewise produced according to the present invention using gus staining . maturation of abies nordmanniana cell lines comprising an auxin phase a ) maturation on culture medium comprising 40 μm aba and 5 % peg - 4000 ( control ). b ) as a ) but culture medium comprising 40 μm aba and 25 μm pcib from week 2 to week 4 . c ) as b ) but culture medium comprising 40 μm aba and 5 μm iaa from week four . embryos produced according to protocol c were longer than embryos produced according to protocol b . the overall quality of these embryos was also higher than the quality of the embryos according to protocol b . maturation of abies nordmanniana cell lines comprising a shift from maltose to sucrose as carbohydrate source during the last period ( the third phase ) of the maturation period except from the carbohydrates , the basic maturation medium was composed as described for the control in example 1 . however , pcib was included in a prior determined optimum concentration and period for each of the tested cell lines . four treatments with different combinations of carbohydrates were tested during maturation : a ) maturation on culture medium comprising 45 g / l sucrose as carbohydrate source during the entire maturation period . b ) maturation on culture medium comprising 45 g / l maltose as carbohydrate source during the entire maturation period . typical embryo from the treatment is disclosed in the left picture of fig9 . c ) maturation on culture medium with 45 g / l maltose during the first part of the maturation period , followed by a shift to maturation medium comprising 22 . 5 g / l sucrose and 22 . 5 g / l maltose during the last two weeks of the maturation period . typical embryo from the treatment is disclosed in the right picture of fig9 . d ) maturation on culture medium with 45 g / l maltose during the first part of the maturation period , followed by a shift to maturation medium comprising 45 g / l sucrose as carbohydrate source for the last two weeks of the maturation period . typical embryo from the treatment is disclosed in the center picture of fig9 . when sucrose was used as carbohydrate source during the entire maturation period ( treatment a ), no well developed mature embryos were produced . all produced mature embryos showed pronounced abnormalities . when maltose was used as the sole carbohydrate source during the entire maturation period ( treatment b ), high numbers of embryos were produced ( as in fig1 c and 2 b ). the mature embryos were well shaped , but for most cell lines the embryos were small compared to mature zygotic embryos . when the cells were transferred from maturation medium with maltose to a maturation medium including sucrose during the last two weeks of maturation ( treatment c and d ), a clear increase in the size of the mature embryos was observed ( fig9 ). a shift to medium including 45 g / l of sucrose ( treatment d ) resulted in the development of mature embryos with a size resembling the size of zygotic embryos of abies nordmanniana . the average fresh weight of mature embryos from treatment b ) was about 50 mg compared to an average fresh weight of mature embryos from treatment d ) of about 120 mg per embryo . an additional effect of treatment d ) compared to treatment b ) was , that the maturation period was reduced with about four weeks . the observed effect of a shift from maltose to sucrose during the last part of the maturation period was surprising and unexpected , because prior investigations has reported a clear negative effect of sucrose during maturation of somatic embryos from abies nordmanniana ( plant science vol 124 : 211 - 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