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Stable Transformation of Using Suspension Cells and Callus

Gladiolus

Kathryn Kamo 1 U.S. Department of Agriculture, Agricultural Research Service, Floral and Nursery Plants Research Unit, Beltsville, MD 20705-2350 Alan Blowers, Franzine Smith, and Joyce Van Eck Sanford Scientific, 877 Marshall Rd, Waterloo, NY 13165 Roger Lawson U.S. Department of Agriculture, Agricultural Research Service, Floral and Nursery Plants Research Unit, Beltsville, MD 20705-2350
Additional index words. transgenic plants, particle gun, GUS
Abstract. More than 100 transgenic Gladiolus plants were recovered after particle bombardment of regenerable suspension cells and callus. For transformation, Glad io lu s callus and suspension cells were co-bombarded with phosphinothricin acetyltransferase-(PAT) and - glucuronidase (GUS) -expressing plasmids. Stably transformed calli were selected on medium containing either phosphinothricin (PPT) or bialaphos followed by transfer to a regeneration medium to recover transgenic plants. Stable transformation was confirmed by detection of the PAT gene by DNA gel blot analysis and by enzymatic assays to measure GUS activity. In general, particle bombardment of regenerable suspension cells rather than callus resulted in the largest number of transformants. The rate of co-expression for GUS in PPT-resistant plants was high 70%). Promoters that are typically more efficient in dicotyledonous plants were very active in Gladiolus, a monocotyledonous bulb plant. Establishment of an efficient transformation protocol for Gladiolus will now allow the introduction of transgenes to confer resistance to the viral and fungal pathogens that decrease Gladiolus production.

Many of the floral bulb crops-tulips, lilies, hyacinths, narcissus, Crocus, Hemerocallis, Iris- are monocots. Before the development of the particle gun (Klein et al., 1987), monocots had not been readil y amenable to transformation using existing techniques. Recently, transgenic plants from severa l agronomically important monocots-corn, wheat, rice, oat, and sugarcanehave been transformed using particl e bombardment of either regenerable suspension cells or callus (Bower and Birch, 1992; Christou et al., 1991; Fromm et al., 1990; GordonKamm et al., 1990; Somers et al., 1992; Vasil et al., 1992; Week s et al., 1993). Gladiolus is a monocotylcdonous floral bulb crop that ranked fifth in 1993 (U.S. Dept. of Agriculture, 1994) for the number of stems (79,663 million) shipped worldwide. Gladiolus production areas are severely plagued by viral and fungal pathogens ; a typical cultivar lasts only several years before it succumbs to disease and is removed from production . Therefore, Gladiolus represents a commercially importan t floral crop that would benefit tremen- dousl y from transgene-mediated crop protectio n strategies. Al- though the infection of Gladiolus cormel tissue by Agrobacterium was reported, definitive evidence for transformation by DNA gel blot analysi s was not presented and no transgenic plants were recovered (Graves and Goldman, 1987). The goal of this study was to produce a large number of transgenic Gladiolus plants. This is the first report of generation of a large number of transgenic plants of a monocotyledonous floral bulb crop.

of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement soley to indicate this fact. To whom reprint requests should be addressed.
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Received for publication 14 June 1994. Accepted for publication 24 Oct. 1994. Mention of a trademark, proprietary product, or vendor does not imply its approval to the exclusion of other products or vendors that may also be suitable. We thank Lisa Lee for advice throughout this study. John Sanford for use of his particle gun, and Anne OConnor and Katerina Serlemitsos for technical assistance. The cost of publishing this paper was defrayed in part by the payment

Materials and Methods Tissue culture. Regenerablc callus was initiate d from either cormel slices or in vitro-grown plantlets of Gladiolus Jenny Lee and Peter Pears (Kamo et al., 1990; Logan and Zettler, 1985) and maintained in vitro for to 6 months on MS basal salts medium (Murashige and Skoog, 1962) supplemented with 2% (w/v) sucrose, 0.2% (w/v) Gelrite (Sigma Chemical Co., St. Louis) and the following (mgliter1): glycine, 1.0; thiamine. 1.0: pyridoxine, 0.5: nicotinic acid, 0.5; either 3,6-dichloro-2-methoxybenzoic acid (dicamba), 2.0; napthaleneacctic acid (NAA). 10.0. or 2,4dichlorophenoxy acetic acid (2,4-D), 0.5, pH 5.8. All media were autoclave at 121C/18 psi. Suspensio n cells were initiated from the regenerable callus in MS basal salts medium supplemented with the same medium components as for callus, except that Gelrite was omitted. Peter Pears and Jenny Lee suspension cells wet-c cultured in 20 ml of MS basal salts medium supplemented with 2 mgliter1 dicamba anti 0.5 mgliter-1 2,4-D, respectively. Callus and suspension cells were grown in the dark at 26C. Callus was routinely transferred every month and suspension cells every 7 days at a 1:2 dilution to fresh medium. Suspension ceils were shaken on a gyratory shaker at 120 rpm/26C in the dark. Plasmid constructs. Plamlid DNA was isolated using alkaline lysis followed by purification from a cesium chloride gradient (Maniatis et al., 1982). Plasmi d p35SAc that contains the PAT (phosphinothricin acetyltransferase) gene under control of the CaMV 35S (cauliflower mosaic virus) promoter (P. Eckes, Hoechst Roussell Co., Somerville. N. Y.) was used to transform plant cells. Plasmids used for the evaluatio n of promoters consisted of the gusA gene under control of the following promoters: Act1 (actin) (McElroy et al., 1991; Zhang et al., 1991), CaMV 35S (Odell et al.. 1985), Ubi1 (ubiquitin) (Christensen et al., 1992), mas2 (mannopine synthase) (Velten et al., 1984), rolD (Leac h and Aoyagi. 1991). All constructs that contained the gusA gene under the various promoters were in either pUC or pUC-like vectors to eliminate any

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possible effect from the vector on gene expression . Plasmids used for evaluation of either the Adh1 (alcoho l dehydrogenase) or Act1 first introns were unde r contro l of the CaMV 35S promoter ((Dennis et al., 1984; McElroy et al., 1991). Particle gun bombardment. The cells were transforme d using the PDS-1000/He system (BioRad , Richmond, Calif.), which is a helium-driven particl e accelerator with flying membrane for particle delivery (Sanford et al.. 1991). M10 tungsten particles were coated with plasmid DNA as previously describe d (Sanford et al., 1993). For all experiments, the gap distance and flying membrane flight distance were set at 1 cm and the target distance at 12 cm. The suspension cells and callus were bombarded at 8.3 MPa (1200 psi) one and two times per plate, respectively. Callus selection. One week following particl e bombardment. the calluS was transferred to MS basal salts medium supplemented with either PPT (phosphinothricin; Hoechst Roussell Co., Somerville . N.J.) or bialaphos (Meiji Seika Kaisha, Tokyo) and either 2 or 1 mgliter dicamba for callus derived from in vitrogrown cormel slices or plantlets, respectively . PPT and bialaphos were added by filter sterilization to autoclaved MS basal salts medium. and the concentration of the selection chemical was based on active ingredient. Callus was transferred every 14 to 21 days to fresh medium during selection containing 6 mgliter-1 P P T or bialaphos. When of the callus had died, the callus was tl-anslerred to regeneration medium , which was MS basal salts medium supplemented with 2 mglite r1 kinetin (Logan and Zettler, 19X5 ) and either- 0, 0.5, or 3.0 mglite r1 bialaphos . Regenerating cultures were grown at 26C under a 16-h photoperiod at 75 E/m2 per sec provided by cool-white fluorescen t lights. Suspension selection. Several methods of selectio n were used for the suspension cells, and all resulted in transgenic plants. For some bombardments, suspensio n cells were transferred to MS basal salts mediu m supplemented with 0.5 mgliter-1 2,4-D and 0.125 M osmoticum (0.0625 M mannitol and 0.0625 M sorbitol) 2 hours he fore particle bombardment; for other bombardments the osmoticum pretreatment was omitted . Following bombardment, the suspension cells were cultured for one week on solidified MS basal salts medium supplemented with 0.5 mglite r1 2.4-D and either with or without 0.125 M ostmoticum to allow the cellS t o recover from bombardment. These cellS were then transferred to solidif ie d MS basa l salt s mediu m supplemente d wit h 0. 5 mgliter 1 2,4-D and either 1 or 6 mglite r1 PPT or bialaphos . After at least two subcultures onto solidified MS basal salts medium supplemented with 2 or 6 mglite r1 bialaphos or PPT, the cel l S were transferred to regeneration medium supple - mented with either 0, 0.5, or 3 mglite r1 bialaphos. A l t e r n a - tively, some of the bombarded cells were cultured 2 weeks in liquid MS basal salts medium supplemented with 0.5 mgliter1 2,4- D and 0.1 mgliter 1 bialaphos immediately afte r bombard- ment. These cells were then transferred to solidified MS basal

salts medium supplemented with 0.5 mgliter -1 2,4- D and 1 mgliter 1 bialaphos until they were ready for transfer to regenera- tion medium. All cells on selection media were transferred every 14 to 21 days to fresh media and grown at 26C in the dark. Cells on regeneration medium were grown in a 16-h light photoperiod at 26C. Shoots that regenerated from the callus and suspension cells were excised and grown on a MS basal salts medium lacking hormone and selection agent where they formed roots. GUS analysis. The method of Jefferson et al. (1987) was used for histochemical and fluorometri c determinations of -glucuronidasc (GUS) gene expression in suspension cells and putative, transformed tissues. The specific activity of GUS was measured by fluorometric determination of 4-methylumbelliferon e (4-MU). The protein concentrations were determined using the BCA protein assay reagent according to the manufacturers instructions (Pierce Co., Rockford , Ill.). Transient GUS expression analysis was used to optimize gene delivery into Jenny Lee suspension cell S that had been bombarded with gu.sA-containin g plasmids. Each experiment consisted of three replicates , and each replicate consisted of two to three plates of suspension cells per treatment. GUS expression was determined 48 h after particle gun bombardment by adding the substrate 5-bromo-3-chloro-3-indolyl -Dglucuronic acid directly to the filter paper on which the suspension cells were cultured. Following a 16-h incubation of the samples with substrate at 37C, the blue spots were counted. Alternatively, the suspension cells were removed from the filter paper, ground up using mortar and pestle, and then used for the fluorometric determination of specific activity of GUS. Nontransformed plant tissues and suspension cells of Jenny Lee and Peter Pears were used as the negativ e controls. DNA analysis. DNA for polymerase chain reactio n (PCR) amplification was isolated from fresh leaves grown in vitro essentially according to the method of Dellaporta et al. (1983). PCR amplification was performed using an Eppendorff microcycler. The PAT gene was amplified using the primer sequence: 5 1 G A G A G G A G A C C A G T T G A G - 3 1 an d 5 ' - T T G G A G G A G C TGGCAAC-3 and the microcycler conditions: 94C for 3 min (one cycle), 94C for 1 min, 55C for 1 min, 72C for 2 min (40 cycles), 72C for 5 min (one cycle). DNA for Southern hybridization was isolated from leaves of in vitro-grown plants using the method of Dellaporta et al. (1983). Undigested DNA (10 g) and DNA digested with either EcoRI or PstI wer e electrophoresed on a 0.7% (w/v) agarose (BioRad, Richmond, Calif.) gel in TBE (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA, pH 8) buffer at 75 V, 50 mA for h. Following electrophoresis, the DNA was transferred by capillary movement ova-night to Nytran nylon membrane (Schleicher and Schuell, Keene, N. Y.) (Maniatis et al., 1982). The probe used for hybridization was p35SAc digested with SalI followed by gel purification 32 and labeled by random primer P] dCTP using the Megaprime

Table 1. Comparison of gene promoter activities in suspension cells of Gladiolus Jenny Lee. Promoter 35S mas2 Gene source CaMV Agrobacterium GUS specific activityz (pmoles 4-MU/h/m g protein) 92 185 Relative GUS expression y 1.00 2.01

rol D Act1 Ubi1

z

Agrobacterium Rice Maize

99 20 28

1 .0 8 0.22 0.30

GUS expression waS measured 48 h after particle gun bombardment of the cells. Values represent the mean of three bombardments (replicates). y Values represnt relative GUS expression, where 35SGUS is set at 1.00.

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Kit (Amersham, Arlington Heights, Ill.). Blots were incubated in prehybridization buffer for 2 h at 42C followed by hybridization buffer and probe at 60C for 16 h (Maniatis et al., 1982). Three washes in 6x SSC, 1% SDS for 5 min each at 26C, two washes in lx SSC, 1% SDS for 1 min each at 65C, and two washes in 2x SSC for 5 min each at 26C were done before exposure of the blot at 70C with an intensifying screen. Results and Discussion Transient GUS gene expression analysis. Initially two promoters, the 35S RNA promoter from cauliflower mosaic virus (CaMV) and the actin 1 (Act1) promote r from rice, fused to gusA w e r e analyzed. The 35S and Act1 promoters have been previously demonstrated to direct high levels of gene expression in transformed dicots and monocots, respectively (McElroy et al., 1990; Zhang et al., 1991). We expected that GUS expression in Gladiolu s would be highest from the Act1 promoter since this promoter is most active in monocots while the CaMV 35S promoter is relatively inactive in monocots . Surprisingly, transient GUS expression levels in bombarded Gladiolus cells were nearly 5-fold higher from the CaMV 35S, promoter than from the Act1 promoter (Table 1). Three additional promoters were evaluated: mas 2 and rol D from Agrobacterium and ubiquitin Ubi1 from maize. From previ- ous reports, the mas2 and rolD promoters would be expected to be most active in dicots wherea s the Ubi1 promoter would be ex- pected to be most efficient in monocots (Christensen et al., 1992; Cornejo et al., 1993; Leach and Aoyagi, 1991; Velten et al., 1984). GUS expression in Gladiolus was highest from the three dicot-

preferred promoters: mas2, rolD, and 35S. We observed that GUS expression from the most efficient promoter, mas2, was 7-fold and 9-fold higher than the Ubi1 and Act1 promoters , respectively. Next we looked at the effect that the presence of monocot introns might have on GUS expression levels since the Ubi1 and Act1 promoter-containin g fragment s used here also contain an intron in their untranslate d leader region (Fig. 1). It is well established that addition of an intron such as the first introns from the maize Adh1 and rice Act1 genes to the primary transcript can dramatically increase gene expression levels in transformed monocots (McElroy et al., 1991). For example, GUS expression in transformed maize cells increased 6- and 60-fold when the Act 1 and Adh1 first introns, respectively, were added to a 35S- gus A chimeric gene. In contrast to maize, GUS expression was reduced 1.25- and 3-fold in Gladiolus when the Adh1 and Act1 first introns, respectively, were added to a 35S- gusA chimeric gene (data not shown). Results similar to those with Gladiolus were found when petunia was bombarde d with the exact same intron-containing plasmids (data not shown). These results clearl y indicated that GUS expression was not stimulated in Gladiolus cells with the addition of monocot introns to the gusA transcript. This observation suggested that splicing of monocot introns may be relatively inefficient in Gladiolus, a nongraminaceous monocot, as it is in dicots such as petunia. Alternatively, stimulation in gene expression by introns may be a feature that is generally limited to graminaceous monocots like maize and rice. Results similar to those obtained with Gladiolu s were also observed with orchid, anothe r nongraminaceous monocot (data not shown). The 35S promoter was most active in bombarded

Fig. 1. Schematic diagrams of the gusA chimeric genes and p35SAc. (Top to bottom) 35S RNA promoter from cauliflower mosaic virus, the actin 5' region that contains the promoter and first intron, the mas2 promoter, the rolD promoter, and the Ubi1 5' region that contains the promoter and first intron. The bottom construct

(p35SAc) is the PAT gene under control of the CaMV 35S promoter. J. AMER . SOC . HORT . SCI. 120(2):347352. 1995.

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orchid protocorms followed by the mas2 promoter . Our results suggest that some nongraminaceou s ornamental monocots should be treated more like dicots rather than monocots when issues regarding transgen e expression are being addressed. It should be recognized that the relative efticiencies of the promoters in regenerate d Gladiolus plants may be different from the values obtained in our transient assays, and this will be addresse d when transgenic plants containing these gusA gene fusions are available for analysis. Others (Russell et al., 1992; Vain et al., 1993) have reported that tobacco and maize suspension cells preconditioned in osmoticum before particl e bombardmen t increased levels of GUS expression 3-fold compared to cells not grown in osmoticum. The optimum concentration of osmoticum resulting in a 3.2-fol d increase of GUS expression was 0.125 M for suspensio n cells of Gladiolus compared to 0.25 M and 0.4 M for tobacco and maize suspension cells, respectively (dat a not shown) . Recovery of transgenic Gladiolus cells. The plasmid , pAct1F4, which contains an Act1- GUS chimeric gene, was co-bombarded with p35SAc DNA into nonregenerabl e Gladiolus cells. For selection, bombarded cells were moved to MS basal salts medium containing either PPT or bialaphos. After 4 weeks, healthy, yellow microcall i began to appear against a background of dead cells. The microcalli were subculture to PPT-containin g medium for continued growth. When PPT-resistan t Gladiolus cells were assayed for GUS activity (Fig. 2A), of the transformants that contained the PAT gene as determined by PCR analysis expressed detectable levels of GUS enzyme. This co-expression rate compares favorably with the rates observed in other plant transformation systems after co-bombardment with two plasmids. Suspension cells of Jenny Lee expresse d high levels of GUS (25 to 40 nmol MU/h per mg protein), which is comparable to the highest levels reported for other monocots (Hagio et al., 1991; Lyznik et al., 1989). Recovery of transgenic Gladiolus plants. Stably transformed Gladiolus plants were recovered followin g particle gun bombardment of either regenerabl e suspension cells or callus with plasmid p35SAc that had the 35 S-PAT chimeric gene to confer resistance to PPT. Only a few of the regenerable cells were co-bombarded with the Act1 GUS chimeric gene resulting in a few plantlets just beginning to differentiate that showed GUS expression (Fig. 2B). Currently, we are in the pocess of transforming plants with the GUS gene under various promoters that will allow us to distinguish between tissue-specific GUS expression and possible chimeras. These results will be reported once these plants are available. To recover transgenic plants (Fig. 2C), PPT-resistant calli that had been bombarded with p35SAc were transferred to regeneration medium. The regeneration medium either lacked the selection agent or contained a lower concentration (0.5 or 3.0 mglite r1 PPT). After 3 to 4 weeks, green shoots appeared on some of the calli. The shoots were transferred to MS basal salts medium lacking hormones and a selectio n agent for further plant development. When multipl e shoots were recovered from a single callus, PCR analysi s performed on individual shoots sampled from the single callus verified that at least 90% of the shoots derived from that callus were transgenic. Twenty-five of the transgenic plants were grown for 2 months on MS medium supplemented with 4.0 mgliter 1 PPT compare d to the nontransformed plants that died after only 1 week on this concentration of PPT, indicating expres- sion of phosphinothricin acetyltransferase by the transgeni c plants. A total of 33 plates of regenerable callus and 18 plates of regenerable suspension cells was bombarded with the PAT gene, resulting in 110 transformed plant s recovered. The 110 transformed plants contain the PAT gene as indicated by PCR analysis.

Fig. 2. (A) Jenny Lee callus showing GUS expression after incubation with 5bromo-4-chloro-3-indolyl-3- -glucuronic acid (X-gluc). The callus had been selected on MS basal salts medium supplemented with 0.5 mgliter 2,4-D and 6 mgliter PPT. (B) Plantlets just beginning to develop and stained to show GUS expression. (C) Transgeni plants of Gladiolus Jenny Lee grown in c vitro on MS basal salts medium without hormones.
l 1

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Fig. 3. DNA blot of genomic DNA isolated from leaves of Gladiolus grown in vitro probed with p35SAc digested with Sal L Each lane contains 10 g DNA. Lane 1: p35SAc digested with EcoRI, which released 50 pg of a 1.34 kb insert. Lane 2: 4 g lambda DNA digested with HindIII. Lanes 3-6 are uncut genomic DNA for nontransformed (NT) Gladiolus or three transformants (T1-3). Lanes 710 are genomic DNA digested with EcoRI for NT or three transformants (T1-3). Size markers shown on the left represent 23.1, 9.4, 6.7, 4.4, 2.3, 2.0, and 0.56 kb, from top to bottom.

Additional putative transformed plants could have been recovered from the selection medium; however, we did not wish to maintain more plants. Southern hybridizations have been performed on nine of the transformants, and two were found to be duplicates. Cur- rently, the other transformed plants are being analyzed by DNA blot analysis so that it can be determined whether they are indepen- dent transformants. DNA gel blot analysis indicated stable incorporatio n of the PAT gene into the Gladiolu s genome (Fig. 3). When the transgenic plants reache d sufficient size, genomic DNA was isolated for DNA gel blot analysis. Genomic DNA samples were digested with EcoRI and probed-with radiolabeled PAT DNA. The expected 1.3 kb EcoRI fragment was observed in lanes containing DNA from the transgenic plants. T3 contains one or only a few copies of the PAT gene compared to T1 and T2. PCR analysis clearly verified the presence of the PAT gene in T3 (data not shown). The amount of DNA loaded onto the gel was identical as that used for T1 and T2, so the difference in signal of the diagnostic band can be attributed to a difference in copy number. The particle gun typi- cally results in a variable number of copies inserted into the plant genome (GordonKamm et al., 1990; Somers et al., 1992; Weeks et al., 1993). No 1.3 kb signal was evident in the lane containing genomic DNA isolated from untransformed plants. There was a difference in the minor bands for T1 and T2, indicating a different pattern of gene rearrangement for each transformant. The particle gun frequently results in gene rearragement s (GordonKamm et al., 1990; Somers et al., 1992; Week s et al., 1993). Additional evidence that T1 and T2 represent independent transformants was provided following digestion of their genomic DNA with PstI that digests once within the transgene (Fig. 4)

Fig. 4. DNA blot of genomic DNA isolated from leaves of Gladiolus grown in vitro probed with p35SAc digested with SalI. Each lane contains 10 g DNA. Lane 1: p35SAc digested with SalI, which released 50 pg of a 559 bp insert. Lane 2:4 g lambda DNA digested with HindIII. Lane 3:2 g 1 kb ladder. Lane 4 genomic DNA digested with PstI from NT plant. Lanes 5 and 6: Genomic DNA digested with PstI from T1 and T2, respectively.

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J. AMER. SOC. HORT. SCI.

1995.

1 Nhm 7:Nui cy huyn ph t bo Bn thn mi t bo thc vt l mt n v c lp, n cha ng tt c nhng thng tin di truyn c trng ca c th t n sinh ra. Cho nn mi t bo c th xy dng li ton b c th mi nh tnh ton nng. Thc vt bc cao la mt ngun cung cp cac hp cht hoa hoc va dc liu rt quan trong. Tuy nhin trong nhng nm gn y san lng cac thc vt o rt kho am bao mc n nh do hu qua cua mt s yu t nh: - iu kin t nhin khng thun li. - Chi phi lao ng ngay cang tng. - Kho khn ky thut va kinh t trong trng trot. Phng phap nui cy huyn ph t bao (dch lng) ca thc vt co kha nng gop phn giai quyt nhng kho khn trn. 2 NI DUNG Khi nim nui cy huyn ph t bo Lch s nghin cu Cc phng php nui cy c trng ca t bo nui cy huyn ph Cc ch tiu xc nh tc sinh trng ng dng ca nui cy huyn ph t bo Cng trnh nghin cu c th Kt lun 3 I. KHI NIM Nui cy huyn phu t bo (cell suspension cultures): Phng thc nui t bao n (single cell) hay cum nhiu t bao (cell aggregate) trang thai l lng trong mi trng long. Dch huyn ph c to ra do s nui cy mt mnh m so khng c kh nng bit ha, trong mi trng lng v c chuyn ng trong sut thi gian nui cy. 4 Cc t bo tch ra khi m so v phn tn trong mi trng lng. Sau mt thi gian, dch huyn ph l mt hn hp cc t bo n, cc cm t bo, cc mnh cn li ca mu cy v cc t bo cht. I. KHI NIM 5 1949: Caplin v Steward nui cy t bo thc vt trn mi trng lng c khuy. Muis (1954) v Nickell (1956) cho thy rng cc t bo thc vt c th sinh trng c nh cc c th vi sinh vt. 1959, Melches v Beckman tch v nui cy thnh cng t bo n v huyn ph t bo. II. LCH S NGHIN CU 6 Nui cy huyn ph t bo Khoai ty 7 III. CC PHNG PHP NUI CY Ty iu kin v mc ch m c cc phng php nui cy t bo huyn ph khc nhau. Ch yu l phng php: Nui cy dch th ng. Trong nui cy dch th ng c h thng cung cp kh (thi kh), cc kh u c v trng. 8 3.1. Nui cy dch th ng 3.1.1. Nui cy chm lin tc Cc t bo c tip xc vi mi trng dinh dng do chng c ngm hn vo dung dch

mi trng. S thng kh c thc hin nh my lc chy tc 100 - 150 vng/pht. Kh a vo phi m bo v trng. Qu trnh thng kh cn ngn chn v lm gim s kt dnh ca cc t bo vi nhau. III. CC PHNG PHP NUI CY 9 III. CC PHNG PHP NUI CY Theo Thomas v Davey (1975), nui cy huyn ph t bo c dung tch 25 ml, tc ph hp nht ca my lc l 100 120 vng/pht. Th tch ca mi trng lng cng phi ph hp vi kch thc bnh nui cy, thng chim 20% th tch bnh. My lc trn 10 Cc nui cy quy m nh v trong nhng thi gian ngn, c th s dng my khuy t tc 250 vng/pht v thi gian cho qu trnh nui cy thng t 10 15 ngy. Sau , cc mu nui cy phi c cy chuyn sang mi trng mi m bo s sinh trng v pht trin ca cc t bo. III. CC PHNG PHP NUI CY 11 3.1.2. Nui cy chm tun hon Cc t bo c nhng chm vo mi trng dch th, xen k vi nhng khong thi gian c a ra ngoi mi trng. Qu trnh c thc hin nh s chuyn ng bp bnh ca cc bnh nui cy. III. CC PHNG PHP NUI CY 12 III. CC PHNG PHP NUI CY Khi chuyn ng: Khi t bo u ny c a vo mi trng. Khi t bo u kia tip xc vi khng kh. Steward v cng s (1952) cng thit k nhng bnh nui cy c bit theo phng php nui cy chm tun hon. H thng nui cy chm tun hon 13 III. CC PHNG PHP NUI CY 3.2. Nghin cu nui cy huyn ph t bo bng Bioreactor: H thng nui cy c thng kh hoc c trao i kh. Mi trng nui cy c th d dng thay mi theo tng thi k. T bo thc vt nhy cm vi stress hn vi khun nn h thng bioreactor cho nui cy t bo thc vt yu cu thng xuyn ci thin h thng thng kh v h thng trn mi trng. T bo thc vt cn nh sng cho quang hp nn cn c s lin kt gia bioreator vi h thng chiu sng. 14 Tng hp lng ln sinh khi to 15 Takayama v Miasawa l nhng ngi u tin s dng bioreactor vo nhn ging cy trng: nhn c siu nh khoai ty, c ging hoa ly, hoa lan h ip. Cng ngh ny cho php nhn nhanh v hn cc ging cy trng nh thit b bioreactor hon ton t ng ha. VD: 1 bioreactor vibro-mixer trang b vi cc ng silicone c kh nng sn xut 100.000 phi v tnh ca cy trng nguyn trong 1lit dch huyn ph nu nh dung dch c t trn 1tm giy lc v pht trin trong 4 tun. III. CC PHNG PHP NUI CY 16

Bioreactor s dng trong nui cy m t bo thc vt c ci tin t cc loi bioreactor trong nui cy t bo vi sinh. III. CC PHNG PHP NUI CY 17 Thun li: Th tch nui cy tng gip sn xut nhiu hn m khng cn nhng k thut cao cp. Hu ht cc bnh bioreactor c thit k vi c ch khuy bng c hc hay thi kh duy tr nui cy gn nh ng dng. III. CC PHNG PHP NUI CY H thng bioreactor nui cy r t nhn sm Hn Quc 18 Khi thao tc nui cy lin tc, mi trng nui cy v mi trng vt l c th c kim sot thch hp cho sinh trng. iu ny khng th thc hin vi h thng nui cy bnh tam gic. Nhc im: i hi thit b hin i v t tin, vn hnh phc tp c bit l khu chng nhim cho huyn ph nui cy. III. CC PHNG PHP NUI CY 19 Bioreactor trong phng Nui cy m t bo v cng ngh t bo thc vt -Vin DT nng nghip 20 Nui cy huyn ph t bo cn mt lng m so kh ln, xp x 2 3 g/100ml dung dch mi trng (Helgeson, 1979). Sau mt thi gian, dch huyn ph l mt hn hp cc t bo n, cc cm t bo, cc mnh cn li ca mu cy v cc t bo cht.

IV. C TRNG CA T BO TRONG NUI CY HUYN PH 21 Mc tch ri ca cc t bo ph thuc vo c tnh ca cc khi t bo xp v c th c iu chnh bi thnh phn mi trng. VD: trong nhiu trng hp, tng t l Auxin/Cytokinin s sn sinh nhiu khi t bo xp. Theo King v Street (1977), khng c mt quy trnh chun no cho nui cy huyn ph t bo. IV. C TRNG CA T BO TRONG NUI CY HUYN PH 22 Sinh trng v pht trin ca t bo nui cy: Pha lag: bt u khi a m so vo mi trng, ko di cho ti ln phn bo u tin. Pha tng tc: cc t bo bt u phn chia v s lng t bo tng dn. Pha hm s m: s lng t bo tng theo hm s m. Pha n nh: s lng t bo t cc i v khng i theo thi gian, s t bo sinh ra (do phn bo) xp x s t bo cht i. IV. C TRNG CA T BO TRONG NUI CY HUYN PH 23 IV. C TRNG CA T BO TRONG NUI CY HUYN PH 24 duy tr qu trnh nui cy, cc t bo cn c cy truyn vo giai on sm ca pha n nh. Thi im cy truyn c th da vo kinh nghim, ni chung l nn bt u khi mt t bo

cc i. Mt t bo cc i trong khong 18 25 ngy, vi huyn ph sinh trng mnh c th ngn hn, t 6 9 ngy. IV. C TRNG CA T BO TRONG NUI CY HUYN PH 25 ln cy truyn u tin, dch nui cn c lc nhm loi b cm t bo ln, cc mnh t mu cy ban u, sau dng pipet ly dch cy truyn. Lng t bo em cy truyn phi ln m bo mt t bo, v khi thp qu cc t bo s khng sinh trng c. VD: i vi t bo cy sung du (Acer pseudoplatanus) mt thch hp 9 15.103 tb/ml. IV. C TRNG CA T BO TRONG NUI CY HUYN PH 26 IV. C TRNG CA T BO TRONG NUI CY HUYN PH Theo King (1980), nhng t bo tri qua qu trnh nui cy, sinh trng v trao i cht trong dch huyn ph gi l dng t bo. c im ca dng t bo: Kh nng tch t bo cao. Hnh thi t bo ng nht. Nhn r rng v t bo cht m c. Nhiu ht tinh bt. 27 IV. C TRNG CA T BO TRONG NUI CY HUYN PH Tng i t cc yu t mch. C kh nng nhn i trong 24 72h. Mt tnh ton nng. Quen vi cht sinh trng. Tng mc a bi th. T bo trong nui cy huyn ph 28 5.1. S lng t bo Trc khi m, x l nhng cm t bo qua acid chromic, un nng 700C trong 5 10 pht, sau lm ngui v lc mnh trong vi pht. Pha long dch, nhum v m trn bung m hng cu. Kt qu: s lng t bo/ml dung dch nui cy. V. CC CH TIU XC NH TC SINH TRNG 29 5.2. Th tch t bo Ly ngu nhin mt th tch dch nui cy, em ly tm tc 2000 vng/pht trong thi gian 5 pht. Thu t bo v em xc nh th tch. Kt qu: S ml t bo/th tch mi trng nui cy. T l %. V. CC CH TIU XC NH TC SINH TRNG 30 5.3. Khi lng ti v khi lng kh t bo Khi lng ti: Thu thp t bo trong mt th tch dch xc nh. Ra bng nc ct v trng Lm kh trong chn khng. Cn xc nh khi lng. Khi lng kh:

Ly mt th tch mu xc nh. Loi b phn ni, ra phn t bo trn giy lc Whatman. Sy kh trong 12h 800C n khi lng khng i. V. CC CH TIU XC NH TC SINH TRNG 31 5.4. Xc nh thnh phn Protein Thu thp t bo, chuyn ln lc qua giy Whatman. Ra t bo trong ethanol 70% ang si. Lm kh vi Aceton ri chuyn vo dung dch NaOH 1M. un nng n 850C trong 1.5h. Lc v xc nh Protein thy phn trong dch theo phng php Lowry v cs (1951). V. CC CH TIU XC NH TC SINH TRNG 32 5.5. Xc nh ch s nguyn phn Huyn ph t bo c c nh trong hn hp Aceto-orcein : Acid Acetic (3:1) sau chuyn sang lam knh. Nh 1 git Aceto-orcein ln mu, h trn ngn la n cn, ngui trong 5 pht. y mu bng Lamel, lm kh mu, quan st di knh hin vi quang hc vt knh 100. Xc nh cc k trong khong 1000 t bo. T l % cc t bo ang phn bo gi l ch s nguyn phn. V. CC CH TIU XC NH TC SINH TRNG 33 6.1. Sn xut sinh khi t bo v cc hp cht th cp Sn phm ca nui cy huyn ph t bo thng c trng l nhng cht c gi tr cao v cn lng nh. C th chia thnh cc nhm da theo cng dng: Hp cht th cp trong y hc. Hp cht th cp trong ha m phm. Hp cht th cp trong cng nghip. VI. NG DNG 34 Ti sao phi nui cy huyn ph t bo thu sinh khi? Tn ph mi trng v s xi mn di truyn. S cung cp ngun nguyn liu khng u n, khng vng chc, cht lng khng n nh. Nhim bn do thuc tr su, bnh hi, cht phng x hay kim loi nng Nui cy huyn ph t bo khc phc c cc nhc im trn. VI. NG DNG 35 VI. NG DNG Ngoi ra, nui cy huyn ph t bo cn c mt s u im sau: Sn xut mt cch c nh hng, theo quy m cng nghip, khi lng sn phm ln, khng ph thuc vo ma v. T l % khi lng kh cc hp cht th cp cao hn so vi nguyn liu thc vt thu hi trong t nhin. Khng tn nhiu din tch gieo trng. Thu sinh khi t bo ln trong thi gian ngn. 36 Mt s hp cht th cp c sn xut t nui cy huyn ph t bo thc vt: Diosgenin: Tc dng chng vim; cha st, i but, i vng, thp khp, au lng, au dy thn kinh. Vic nui cy Dioscorea deltoidea Wall ex Griseb (cy C nu) trong bioreactor thu c

nng sut diosgenin cao hn so vi hiu sut c c t nguyn liu t nhin. Trong nui cy t bo, diosgenin chim 7.8 % khi lng kh, trong khi ch chim 2 % vi nguyn liu thc vt thu hi trong t nhin. VI. NG DNG 37 VI. NG DNG Codein: Gim au v gim ho. Papaver somniferum l ngun ca 2 hp cht rt quan trng trong y hc l morphine v codein. Sn xut morphin c thc hin bng vic nui cy huyn ph t bo t bo Papaver somniferum. Cy anh tc (Papaver somniferum) 38 VI. NG DNG Steviol: Cht to v ngt c bit n rng ri, thu nhn t cy Stevia rebaudiana. Nui cy callus l giai on tin nui cy huyn ph t bo trong tch ly steviol. Trong nui cy callus, hiu sut t ti 36.4 %. Cy c ngt (Stevia rebaudiana) 39 VI. NG DNG Taxol: - L mt nhm cht trong ha tr liu ung th, iu tr ung th bung trng, ung th v, ung th phi, khi u c tnh, v cc dng u bu khc. - Nui cy t bo cc loi Taxus c xem nh l mt phng php u th cung cp n nh ngun taxol v dn xut taxane ca n (T. cuspidata, T. baccata, T. mairei). Cy thng (Taxus brevifolia) 40 VI. NG DNG Scopolamine v hyoscyamine: L 2 loi tropane alkaloid c s dng tr co tht v gy m. C nhiu trong cc cy thuc h Solanaceae. Nui cy huyn ph t bo Scopolia japonica c b sung tropic acid (nh 1 tin cht) th c th tng hm lng alkaloid ln 15 ln. C c dc (Datura metel) 41 VI. NG DNG Saponin v sapogenin: L mt dc phm qu gi, c nhiu trong r cy nhn sm (Panax ginseng), c tc dng cha bnh ri lon tiu ha, chng suy nhc c th, tng cng sinh lc. Nui cy huyn ph t bo cy P. ginseng ang tin hanh trn quy m cng nghip. y l mt trong cc i tng c cc nh khoa hc trn th gii tp trung nghin cu nhiu nht. Nhn sm (Panax ginseng) 42 VI. NG DNG Betalaine: L mt nhm cc cht nhum mu thc phm. Bao gm betaxanthine (mu vng) v betacyane (mu ). Thnh tu nui cy c quan cy Beta vulgaris, nui cy callus v huyn ph ca Portulaca americana. C ci (Beta vulgaris) 43 6.2. Trong cng tc chn ging cy trng: S dng chn lc cc dng t bo mong mun qua cc test thanh lc.

Sau ti sinh cc t bo chn lc thnh cy hon chnh. Cy hon chnh s tr thnh vt liu khi u quan trng trong cng tc ging. Ngoi ra, tch protoplast hoc l ngun sn xut cc phi v tnh. VI. NG DNG 44 45 Nghin cu nui cy m so v dch huyn ph cy Bt rui Drosera indica L. cho mc tiu thu nhn Plumbagin. (Quch Dim Phng). Nghin cu nui cy m so v dch huyn ph cy Bo t Drosera burmalni Vahl cho mc tiu thu nhn 1 hp cht Anthraquinone. Quch Dim Phng ( ti NCCB ca H Quc gia TP HCM) VII. CNG TRNH NGHIN CU 46 Nghin cu nui cy m so v dch huyn ph cy Mn nh Hng Althaea rosea L. nhm thu nhn Rutin trong iu tr tim mch. (Quch Dim Phng). Nghin cu ng dng cng ngh nui cy t bo h cy Drosera.sp thu nhn cc hp cht Quinone c hot tnh sinh hc. (Quch Ng Dim Phng). VII. CNG TRNH NGHIN CU 47 Thnh cng trong quy trnh to sinh khi t bo r sm Ngc Linh bng ng dng cng ngh Biomass Hc vin Qun Y. Cc nh khoa hc Vin Cng ngh sinh hc (Vin khoa hc v Cng ngh Vit nam) hon chnh quy trnh nhn nhanh sinh khi m ca cy Hong Lin gai (Berberis Wallichiarna DC) bng cng ngh t bo thc vt lm ngun dc liu sn xut thuc becberin. VII. CNG TRNH NGHIN CU 48 Tuy nhin, do nhng kh khn trong vic tm kim ti liu nn nhm chng em khng a ra c mt quy trnh sn xut da trn nui cy huyn ph t bo, m ch a ra c mt cng trnh nghin cu giai on u: 49 NH HNG CA CHT IU HA SINH TRNG THC VT V NG SACCHAROSE LN DCH NUI CY HUYN PH T BO DA CN CATHARANTHUS ROSEUS Tc gi: Bi Vn L, Trng i hc Khoa hc T nhin, HQG-HCM Nguyn Ngc Hng, Trng i hc Bn cng Tn c Thng VII. CNG TRNH NGHIN CU 50 1. T VN Da cn Catharanthus roseus.G.Don thuc h Trc o Apocynaceae l mt trong nhng dc liu cha nhiu alkaloid. T da cn ngi ta chit c cht cha ung th nh vinblastin v cha cao huyt p nh ajmalicin, serpentin. Tuy nhin hm lng ca nhng cht ny c trong cy l rt thp. Vic nui cy t bo cy da cn nng cao hm lng alkaloid mong mun c nhiu nc trn th gii nghin cu v ng dng vo sn xut. VII. CNG TRNH NGHIN CU 51 Cy da cn Catharanthus roseus 52

Ajmalicin

Vinblastin 53 2. VT LIU V PHNG PHP M so mu xanh c nui trong iu kin chiu sng t l da cn in vitro 4 thng tui c chuyn vo 100 ml mi trng cm ng trong erlen 250 ml. Mi trng cm ng gm mi trng MS + Vitamin Morel + Hormon tng trng thc vt (NAA hoc 2,4-D v Kin hoc BAP) + 30g/l ng sucrose. Sau t vo my lc 200 vng/pht nhit 25 2oC trong iu kin chiu sng lin tc 16 gi/ngy. VII. CNG TRNH NGHIN CU 54 Xc nh sinh khi bng phng php cn. Chit tch alkaloid ton phn t sinh khi t bo Da cn theo phng php ca Kutney v cng s (1983). Xc nh alkaloid ton phn bng phng php acid-baz - khan theo dc in Vit Nam v dc in Anh. VII. CNG TRNH NGHIN CU 55 VII. CNG TRNH NGHIN CU Quy trnh thc hin th nghim 56 3. KT QU V THO LUN 3.1. Kho st nh hng ca s thay i nng cytokinin n s to sinh khi 57 3. KT QU V THO LUN 3.1. Kho st nh hng ca s thay i nng cytokinin n s to sinh khi ng cong tng trng ca dch huyn ph t bo da cn trong mi trng b sung NAA v cytokinin 58 3. KT QU V THO LUN 3.1. Kho st nh hng ca s thay i nng cytokinin n s to sinh khi Kt lun: bng trn cho thy mi trng ch b sung NAA c s to sinh khi thp hn so vi mi trng b sung NAA kt hp vi Kin. Mi trng K4 (MS + NAA (1mg/l) + Kin (0.5 mg/l) v K8 (MS + NAA (1mg/l) + BAP (0.5 mg/l) cho sinh khi ti v kh u cao hn cc mi trng khc.

59 3. KT QU V THO LUN 3.2. nh hng ca cc cht iu ha sinh trng n vic to sinh khi 60 3. KT QU V THO LUN 3.2. nh hng ca cc cht iu ha sinh trng n vic to sinh khi

ng tng trng ca dch huyn ph t bo da cn sau 28 ngy nui trong mi trng c b sung cc hormone thc vt khc nhau. 61 3. KT QU V THO LUN 3.2. nh hng ca cc cht iu ha sinh trng n vic to sinh khi Kt lun: Mi trng c b sung 2,4-D to sinh khi ti nhiu hn so vi mi trng b sung NAA nhng trng lng kh li rt thp. Mi trng K3 v K4 c trng lng ti thp hn mi trng K1 v K2 nhng cho sinh khi kh nhiu hn. Mi trng K3 cho sinh khi ti v kh u cao hn mi trng K3 nhng xt v mt thng k, hai mi trng ny khng c s khc bit mc ngha = 0.01.

62 3. KT QU V THO LUN 3.2. nh hng ca cc cht iu ha sinh trng n vic to sinh khi Kt lun: 2,4-D kch thch s phn chia t bo nhanh nhng li ph hy cy trc cht ch ca t bo lm cho t bo xp v gim i vic to cc sn phm th cp. Cytokinin cn thit cho s hnh thnh cc hp cht th cp, khi kt hp cytokinin vi NAA gip duy tr s tng trng cng nh to ra alkaloid cao. 63 3. KT QU V THO LUN 3.3. nh hng ca ng sucrose n vic to sinh khi 64 3. KT QU V THO LUN 3.3. nh hng ca ng sucrose n vic to sinh khi ng tng trng ca dch huyn ph t bo trong mi trng c b sung cc nng ng khc nhau 65 3. KT QU V THO LUN 3.3. nh hng ca ng sucrose n vic to sinh khi Kt lun: Nng ng tng dn t 20 - 60 gam/l sinh khi ti v kh thu c trong mi trng tng dn theo. Mi trng ti u cho th nghim v nh hng ca nng ng ln s to sinh khi l mi trng 5. Tuy nhin khi tng nng ng ln cao hn l 70 - 80 gam/lt th trng lng ti v kh gim dn. Nng ng c nh hng n s to thnh cc sn phm th cp trong nui cy t bo. nng ng sucrose cao va phi khong 50 60 gam/lt s kch thch to sinh khi v alkaloid.

66 3. KT QU V THO LUN 3.4. nh lng alkaloid vinblastin v vincristin c trong l cy khng qua nui cy in vitro v trong dch huyn ph t bo

67 3. KT QU V THO LUN 3.4. nh lng alkaloid vinblastin v vincristin c trong l cy khng qua nui cy in vitro v trong dch huyn ph t bo Kt lun: bng phng php nui cy dch huyn ph c b sung 2,4-D th lng vincristin v vinblastin khng c trong mu alkaloid ton phn cn dch huyn ph NAA + Kin cho lng vinblastin thp hn so vi cy trng ngoi t nhin nhng cho lng vincristin kh cao trong khi cy trng ngoi t nhin trong th nghim ny li khng c.

68 4. KT LUN: Tm -naphthaleneaceticc nng hormon ph hp: nng 1 mgl-1 acid (NAA) v 0,5 mgl-1 kinetin (Kin) thu nhn c sinh khi v alkaloid ton phn cao nht trong khi cng mi trng ny nhng thay th NAA bng 2,4-dichlorophenoxyacetic acid (2,4-D) cng nng thu c sinh khi v alkaloid ton phn thp. Nng ng ti u cho vic nui dch huyn ph t bo da cn: nng 60 gl-1 cho lng sn phm l cao nht.

VII. CNG TRNH NGHIN CU 69 VIII. KT LUN Vi nhiu u im vt tri ca mnh, phng php nui cy huyn ph t bo ang c p dng rng ri trong nghin cu cng nh trong thc tin sn xut. Nui cy huyn ph t bo ang m ra nhiu nh hng pht trin cho tng lai. 70 Tuy nhin, do c s vt cht c bn cho mt phng th nghim nui cy huyn ph t bo cn cao nn Vit Nam, hng nui cy huyn ph t bo ch dng li mc nghin cu. Trong nhng nm gn y, Vit Nam thnh cng trong nhng quy trnh p dng cc phng php ca nui cy huyn ph t bo sn xut ra cc ch phm sinh hc, y hc VIII. KT LUN 71 Ti liu tham kho Cng ngh sinh hc. Tp 2: Cng ngh t bo. NXB Gio dc. Cc ngun ti liu trn Internet 72

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