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Transgenic wheat plants: the end of the beginning
Transgenic
suitable, howsvcr, fbr dr llvcry of gems 10 callus. The Gaincsvillc group thcrefk turned to bit&tics high-velocity microprojectilc hombardmcnt (secJ. Sandford ?‘iU’I%CH 6, 299-302, 1988) - as the method ofchoicu.
that thorn is sotile n;ltiiral rcaistanco to t!?i* herbicide in wheat cells. Of thl. cxprrimcntal calli which wcrc rcsistant to 13asta’~‘,bcrmcn 6% and 10% cxhlbitcd PAT activity, whcrcas non,: of the hcrbicitlc-resistlIlt control calli exhibited enzyme a, rivity. Those calli which exhibited PAT
The next step The next step was to induce in emb~ogenesis/morphogenesis the transgenic calli Somatic embryos and shoots were formed from calli of all four wheat cultivars, but in only two of thee w(:rc whole plants (dlc rcgcuerated; 70% of IX,, gcnrration) the Ii,, plants cxhibitcd PAT activity. I:, pl.mtr wcrcs ob!aincd by pollinating R,, stigmas with wild-type pollen and by pollinating wild-type stigmas with K,, pollen. Only R, progsny from thil former cross (Ii,,? s W.I. 6) exhibited PAT activity (and CLvctiin this group. only 50%~ of ,ihc plants wc‘rc PAT+). Southern analysis indicated that thrse PAT+ R, plants contained the bar gene, integrated into nuclear DNA. The two transgenic R, plants were used to gcncrate R2 plants by selfpollination and by cross-pollination with wild-type pollen. These pollinations yielded 262 seeds, 6X of which were germinated; 57 of the resulting plants were used to assay for PAT activity and BastG rcsiscance. Thcrc was close correlation between thcsc two characus, indisaeing d,nt both rcsultcd irum possession of the BRr gene. The segregation pattern of Ihc bar gene in die I$ gcncration was 3:l in the progeny from the selfpollination and 1:1 in the progeny from the out-crossing. These data indicate that the bar gcno was
I*It’s
well,
Introducing a gene construct The gcnc cnmxtuct used@ARG’JS), contained a reporter grnc (,crcs, cucoding P-glucuronidasc) m&l th,. cc\ntrrlJ of !I!: .~L*l..q ,*.. u.. I dclqdrogvnasc (Adh)-1 prtrmotcr, :md :I sc:lcctablc marker ~C'IIC, bnr. un&r tlic ~ont:ol dtlx CaM; 35S prcmmtw, Thr hlrr geuo ~IWX&S F~~0s~~bintrthricin acetyl transfcrasc (PAT) which inactivates the hcrbiaide Bnst~‘~~,the active ilqggediLnt of which is phosphinotllricin. Embryogcnic calli from four wheat culti qars were bombarded with microprojcctiles coated with pBARGUS; control calli were bombarded with uncoated microprojecriles. The calli wcrc then grown on a medium containing the hrrhicidc Un+a”‘” for calli bombarded wirh l),V\Ip., the survival rate was between X’S and L?O’%;and for control calli, the survival rate bctwrrn Y% :III~ I%, showinK
the
best --
thing
was
since,
er..,.slicecl
03 1992, Elsev~er SilencePublisherskI (UK)
bread
!”
343 f OYl4tB
inherited mend&n
as a single, trait.
market, but, without doubt, this step forward represents the end of the beginning for transgcnic wheat.
tlominaut
A significant step forward GaincsvG group have The devoted much rffort to the pduction oftransgritic wheat, and deserve the conptulations of the Srotechnology co~umn+y for this success.
It will ofcourse be some time before we see the fruits ofthis success on the
References 1 dhisnmotcr,K., Tursda, R., Izwa. T. and Fujimoto. H. (19RO)Ncr$rw338, 276277 2 Gordon-Karnm, W. J.. Spcnccr.T, M., Mangano, M. L., Adams. T. K., I~aincr, K. J., Stan, W. G., O’Brien, J. V., Chambers,S. A., Adams, W. R., Willets,
Imageanalysis:putting filamentousmicroorganismin the picture Image analysis can be used to characterize
the morphology
Colin R. Thomas
and simple
differentiation of fungi and actinomycetes. lt provides a new and powerful tool for the
physiologist
or fermentation
technologist
working
with filamentous
microorganisms. Many fermentations of industrial importance exploit the metabolism offilamentous microorganisms such as fungi and actinomycctes. Such fermentations yield high-value products, including most antibiotics and somr enzymes and organic acids, in addition to the microbial biomass which may bc used in foodstufi. A fungal or actinomycctc mycelium consists of a net~:srk af branching hyphae, typically only J-10 km in diameter for fung and 0.5-I .5 pm for acS~o:;1-j~~:~;‘, but that may bc up to several hundred microns long. This mode of growth aLows thr organism to incrcdSe in size without altering the protoplasmic volume : surface-arca ratio, whicrl makes thcsc microorganisms well adapted to colonization ofsolid substrates!. Howcvcr. for economical prodxtioo, most processes require submerged culture in large fermenters, often with intense agitation ant! aeration. Under these conditions, a wide variation in gross morphology (shape) is found, varying from discrete or loosely ental:gled filaments (Fig. :,ij, to pellet; (spherical colonies of highly I:ntangled hyphal tmm; Fig. 2a). In many cases, productivity sec~ns to depend on morphology, pa&&r!y f-\r r+ucntations using pelleted organisms2. In addition, the dispencd form of growth (Fig. la) can lead to hyphal entanglement in the fermentation broth. This may have an adverse effect 0;: the rheological properties of the broth, making it highly viscous an?. pseudoplastic, and thus diticult to mix in 0
1992. Elwer SciencePublishers LtdklK)
a large fermenter, so that inhomcpneities might arise. Furtl;ennorc, oxygen tramfer from sparged air bubbles to thr microorganisms decreases with increasing viscoszty. In severe cases, this can lead to oxygen limitation. Heat transfer for fermcntcr cooling can also be reduced under such conditions. These problems can lead to lowered productivity. Such rheological przbicms do not tend :o a%= in fermentations with pclletcd forms, hut the centres of large pellets might be starved of oxygen and autolyse, again affecting productivity. Clearly the mo~hology of these tilameutous microorganisms is a mdttcr of profLu11d concsm for fermsntation scientists. ether types ofstructural variation can also occur in filamcntous microorganisms. Some fungi and a11actinomycctcs are septate (i.e. they have crosswalls which divide the hyphae into a series of compartments). As growth OCCUISonly at the hyphal tips’, &cse hyphnl compartments vary greatly in age and in physiological state, and can show *Jarious forms ofstructural di&rentiation3~J. For e.iatnple, vacuoles can appear in the hyphar during ;ater stages of grflwth (Fig. la). 111 general, the further the compartmr:z is from the apex (i.e. the older the compartment), the larger the vacuoles. The expression of genes associated with secondary metabolic activities such as antibiotic production might be re!atcd to the process of differentiation. Blll.---- --101 ltBTEClf-----“---‘ OCKBER----’ 199Z!VOL
suitable, howsvcr, fbr dr llvcry of gems 10 callus. The Gaincsvillc group thcrefk turned to bit&tics high-velocity microprojectilc hombardmcnt (secJ. Sandford ?‘iU’I%CH 6, 299-302, 1988) - as the method ofchoicu.
that thorn is sotile n;ltiiral rcaistanco to t!?i* herbicide in wheat cells. Of thl. cxprrimcntal calli which wcrc rcsistant to 13asta’~‘,bcrmcn 6% and 10% cxhlbitcd PAT activity, whcrcas non,: of the hcrbicitlc-resistlIlt control calli exhibited enzyme a, rivity. Those calli which exhibited PAT
The next step The next step was to induce in emb~ogenesis/morphogenesis the transgenic calli Somatic embryos and shoots were formed from calli of all four wheat cultivars, but in only two of thee w(:rc whole plants (dlc rcgcuerated; 70% of IX,, gcnrration) the Ii,, plants cxhibitcd PAT activity. I:, pl.mtr wcrcs ob!aincd by pollinating R,, stigmas with wild-type pollen and by pollinating wild-type stigmas with K,, pollen. Only R, progsny from thil former cross (Ii,,? s W.I. 6) exhibited PAT activity (and CLvctiin this group. only 50%~ of ,ihc plants wc‘rc PAT+). Southern analysis indicated that thrse PAT+ R, plants contained the bar gene, integrated into nuclear DNA. The two transgenic R, plants were used to gcncrate R2 plants by selfpollination and by cross-pollination with wild-type pollen. These pollinations yielded 262 seeds, 6X of which were germinated; 57 of the resulting plants were used to assay for PAT activity and BastG rcsiscance. Thcrc was close correlation between thcsc two characus, indisaeing d,nt both rcsultcd irum possession of the BRr gene. The segregation pattern of Ihc bar gene in die I$ gcncration was 3:l in the progeny from the selfpollination and 1:1 in the progeny from the out-crossing. These data indicate that the bar gcno was
I*It’s
well,
Introducing a gene construct The gcnc cnmxtuct used@ARG’JS), contained a reporter grnc (,crcs, cucoding P-glucuronidasc) m&l th,. cc\ntrrlJ of !I!: .~L*l..q ,*.. u.. I dclqdrogvnasc (Adh)-1 prtrmotcr, :md :I sc:lcctablc marker ~C'IIC, bnr. un&r tlic ~ont:ol dtlx CaM; 35S prcmmtw, Thr hlrr geuo ~IWX&S F~~0s~~bintrthricin acetyl transfcrasc (PAT) which inactivates the hcrbiaide Bnst~‘~~,the active ilqggediLnt of which is phosphinotllricin. Embryogcnic calli from four wheat culti qars were bombarded with microprojcctiles coated with pBARGUS; control calli were bombarded with uncoated microprojecriles. The calli wcrc then grown on a medium containing the hrrhicidc Un+a”‘” for calli bombarded wirh l),V\Ip., the survival rate was between X’S and L?O’%;and for control calli, the survival rate bctwrrn Y% :III~ I%, showinK
the
best --
thing
was
since,
er..,.slicecl
03 1992, Elsev~er SilencePublisherskI (UK)
bread
!”
343 f OYl4tB
inherited mend&n
as a single, trait.
market, but, without doubt, this step forward represents the end of the beginning for transgcnic wheat.
tlominaut
A significant step forward GaincsvG group have The devoted much rffort to the pduction oftransgritic wheat, and deserve the conptulations of the Srotechnology co~umn+y for this success.
It will ofcourse be some time before we see the fruits ofthis success on the
References 1 dhisnmotcr,K., Tursda, R., Izwa. T. and Fujimoto. H. (19RO)Ncr$rw338, 276277 2 Gordon-Karnm, W. J.. Spcnccr.T, M., Mangano, M. L., Adams. T. K., I~aincr, K. J., Stan, W. G., O’Brien, J. V., Chambers,S. A., Adams, W. R., Willets,
Imageanalysis:putting filamentousmicroorganismin the picture Image analysis can be used to characterize
the morphology
Colin R. Thomas
and simple
differentiation of fungi and actinomycetes. lt provides a new and powerful tool for the
physiologist
or fermentation
technologist
working
with filamentous
microorganisms. Many fermentations of industrial importance exploit the metabolism offilamentous microorganisms such as fungi and actinomycctes. Such fermentations yield high-value products, including most antibiotics and somr enzymes and organic acids, in addition to the microbial biomass which may bc used in foodstufi. A fungal or actinomycctc mycelium consists of a net~:srk af branching hyphae, typically only J-10 km in diameter for fung and 0.5-I .5 pm for acS~o:;1-j~~:~;‘, but that may bc up to several hundred microns long. This mode of growth aLows thr organism to incrcdSe in size without altering the protoplasmic volume : surface-arca ratio, whicrl makes thcsc microorganisms well adapted to colonization ofsolid substrates!. Howcvcr. for economical prodxtioo, most processes require submerged culture in large fermenters, often with intense agitation ant! aeration. Under these conditions, a wide variation in gross morphology (shape) is found, varying from discrete or loosely ental:gled filaments (Fig. :,ij, to pellet; (spherical colonies of highly I:ntangled hyphal tmm; Fig. 2a). In many cases, productivity sec~ns to depend on morphology, pa&&r!y f-\r r+ucntations using pelleted organisms2. In addition, the dispencd form of growth (Fig. la) can lead to hyphal entanglement in the fermentation broth. This may have an adverse effect 0;: the rheological properties of the broth, making it highly viscous an?. pseudoplastic, and thus diticult to mix in 0
1992. Elwer SciencePublishers LtdklK)
a large fermenter, so that inhomcpneities might arise. Furtl;ennorc, oxygen tramfer from sparged air bubbles to thr microorganisms decreases with increasing viscoszty. In severe cases, this can lead to oxygen limitation. Heat transfer for fermcntcr cooling can also be reduced under such conditions. These problems can lead to lowered productivity. Such rheological przbicms do not tend :o a%= in fermentations with pclletcd forms, hut the centres of large pellets might be starved of oxygen and autolyse, again affecting productivity. Clearly the mo~hology of these tilameutous microorganisms is a mdttcr of profLu11d concsm for fermsntation scientists. ether types ofstructural variation can also occur in filamcntous microorganisms. Some fungi and a11actinomycctcs are septate (i.e. they have crosswalls which divide the hyphae into a series of compartments). As growth OCCUISonly at the hyphal tips’, &cse hyphnl compartments vary greatly in age and in physiological state, and can show *Jarious forms ofstructural di&rentiation3~J. For e.iatnple, vacuoles can appear in the hyphar during ;ater stages of grflwth (Fig. la). 111 general, the further the compartmr:z is from the apex (i.e. the older the compartment), the larger the vacuoles. The expression of genes associated with secondary metabolic activities such as antibiotic production might be re!atcd to the process of differentiation. Blll.---- --101 ltBTEClf-----“---‘ OCKBER----’ 199Z!VOL