CA2383944A1 - Efficient production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (hordeum vulgare l.) genotypes - Google Patents
Efficient production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (hordeum vulgare l.) genotypes Download PDFInfo
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Abstract
The in vitro plant regeneration frequencies for immature scutella, leaf-bases/apical meristems (LB/AM) and mature embryos of four commercially important barley genotypes were compared. Production of shoots from mature embryos or calli of LB/AM incubated on media containing 1.0 or 2.0 mg/L 6-benzyiaminopurine (BAP) were comparable to regeneration frequencies obtained for scutella-derived calli of the same genotypes. Incubation of excised mature embryos and LB/AM on media containing an alternative plant growth regulator, thidiazuron (TDZ), resulted in an increased shoot production. However, TDZ treatment did not stimulate plant regeneration from calli derived from scutella or LB/AM. Shoots formed from TDZ-treated mature embryos and LB/AM were induced without a callus interphase and the in vitro culture system gave a three- to sight-fold higher regeneration frequency than recorded for scutella-derived calli on BAP medium. The simplicity and rapid development of shoots using the mature embryo system strongly favours its use over commonly used regeneration systems.
Description
BACKGROUND OF THE INVENTION
Barley is one of the most recalcitrant cereal crops with only a limited number of tissues suitable for in vitro culture and plant regeneration. Most barley tissue culture and transformation studies have used immature tissues, such as immature embryos (Ritala et al. 1994; Wan and Lemaux 1994) or scutella (Tingay et al. 1997;
Weir et al. 1998). Plant regeneration using these explants generally occurs via somatic embryogenesis, where an embryogenic callus is f rst formed, followed by embryo maturation and production of plantlets. Although mature plants can be routinely regenerated from immature explants of several barley cultivars, the process is to laborious, requires constant growth of donor plants under controlled conditions and is highly genotype-dependent (Liihrs and Lorz 1987). Furthermore, commercially grown barley cultivars are often found to respond poorly to in vitro culture.
More readily available explant sources, such as mature embryos and leaf bases have also been evaluated for in vitro culture. Lup~tto (1984) reported both callus induction and plant regeneration from mature embryos, but low regeneration frequencies did not allow widespread use of the system. Regeneration experiments conducted by Saalbach and Koblitz ( 1978) showed that leaf-base segments from barley seedlings could be induced to produce callus on culture media containing high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Similar to early experiments with mature embryos, regeneration of plants from leaf base-induced callus was a rare occurrence. Mohanty and Ghosh (1988) improved the leaf base tissue culture and reported more efficient production of somatic embryos and plant regeneration. In a study on leaf base tissue culture, Kapur ( 1991 ) suggested that plant regeneration is only possible when the apical meristem is present within the excised explant. However, in a recent study Pasternak et al. (1999) demonstrated efficient production of embryogenic callus and subsequent plant regeneration from young leaf tissue segments devoid of apical meristems.
Isolated barley apical meristems were among the initial explants used in tissue culture and plant regeneration from callus tissue could be achieved via organogenesis (Cheng and Smith 1975) or embryogenesis (Weigel and Hughes 1985). Rengel and Jelaska (1986) modified the apical meristem system by using the basal part of barley seedlings containing the scutella, the apical meriste;m, the youngest leaves and the coleoptiles as explants. These explants initially produced non-embryogenic calli 1o which, upon sub-culture, developed into embryogenic calli followed by plant regeneration. Direct production of multiple shoots without a callus induction phase has also been reported for isolated barley apical meristems (Zhang et al.
1998).
Production of shoots from explant tissues often occurs via a callus phase and 2,4-D is one of the most potent auxins for induction of embryogenic callus.
Further maturation of the embryos and plant development generally requires a culture medium containing cytokinins and a low concentration or no auxins. The plant growth regulator, thidiazuron (TDZ), with both cytokinin- and auxin-like properties has been reported to promote plant regeneration in a number of species by stimulating production of axillary and/or adventitious shoots or somatic embryos (Huetteman and 2o Preece 1993; Lu 1993; Murthy et al. 1998). In cereals, TDZ-containing media have been used to promote shoot induction from wheat (Hong and Zhen 1999; Shan et al.
2000), barley (Shan et al. 2000) and rice (Tian 1994; Azria and Bhalla 2000) calli.
Gupta and Conger (1998) used a combination of TDZ and 2,4-D in the medium to induce multiple shoots directly from intact seedlings of switchgrass, Panicum virgatum. Nodal explants of Bambusa edulis have also been induced to produce multiple shoots in response to TDZ (Lin and Chang 199$). Similarly for sugarcane, calli induced from immature inflorescences produced shoots on a TDZ-containing medium (Gallo-Meagher et al. 2000).
Studies are constantly being conducted to improve callus production and regeneration from barley by identifying explant systems that are more amenable to in vitro culture and improving media formulations.
SUMMARY OF THE INVENTION
1o This invention relates to the determination of regeneration efficiencies of LB/AM and mature embryos as compared to the regeneration frequency from immature scutella, when treated with TDZ.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1, a,b,c are graphs illustrating the effects of culture media composition on regeneration of barley plants.
Figure 2 a,b are graphs illustrating the effect of TD Z on short induction frequencies in barley LB/AM and mature embryos 2o DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods Seeds of CDC Dawn, CDC McGwire, CDC Copeland and BT459 were obtained from the Crop Development Centre, University of Saskatchewan. CDC
Dawn and CDC McGwire are 2-rowed hulless feed barley varieties, whereas CDC
Barley is one of the most recalcitrant cereal crops with only a limited number of tissues suitable for in vitro culture and plant regeneration. Most barley tissue culture and transformation studies have used immature tissues, such as immature embryos (Ritala et al. 1994; Wan and Lemaux 1994) or scutella (Tingay et al. 1997;
Weir et al. 1998). Plant regeneration using these explants generally occurs via somatic embryogenesis, where an embryogenic callus is f rst formed, followed by embryo maturation and production of plantlets. Although mature plants can be routinely regenerated from immature explants of several barley cultivars, the process is to laborious, requires constant growth of donor plants under controlled conditions and is highly genotype-dependent (Liihrs and Lorz 1987). Furthermore, commercially grown barley cultivars are often found to respond poorly to in vitro culture.
More readily available explant sources, such as mature embryos and leaf bases have also been evaluated for in vitro culture. Lup~tto (1984) reported both callus induction and plant regeneration from mature embryos, but low regeneration frequencies did not allow widespread use of the system. Regeneration experiments conducted by Saalbach and Koblitz ( 1978) showed that leaf-base segments from barley seedlings could be induced to produce callus on culture media containing high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Similar to early experiments with mature embryos, regeneration of plants from leaf base-induced callus was a rare occurrence. Mohanty and Ghosh (1988) improved the leaf base tissue culture and reported more efficient production of somatic embryos and plant regeneration. In a study on leaf base tissue culture, Kapur ( 1991 ) suggested that plant regeneration is only possible when the apical meristem is present within the excised explant. However, in a recent study Pasternak et al. (1999) demonstrated efficient production of embryogenic callus and subsequent plant regeneration from young leaf tissue segments devoid of apical meristems.
Isolated barley apical meristems were among the initial explants used in tissue culture and plant regeneration from callus tissue could be achieved via organogenesis (Cheng and Smith 1975) or embryogenesis (Weigel and Hughes 1985). Rengel and Jelaska (1986) modified the apical meristem system by using the basal part of barley seedlings containing the scutella, the apical meriste;m, the youngest leaves and the coleoptiles as explants. These explants initially produced non-embryogenic calli 1o which, upon sub-culture, developed into embryogenic calli followed by plant regeneration. Direct production of multiple shoots without a callus induction phase has also been reported for isolated barley apical meristems (Zhang et al.
1998).
Production of shoots from explant tissues often occurs via a callus phase and 2,4-D is one of the most potent auxins for induction of embryogenic callus.
Further maturation of the embryos and plant development generally requires a culture medium containing cytokinins and a low concentration or no auxins. The plant growth regulator, thidiazuron (TDZ), with both cytokinin- and auxin-like properties has been reported to promote plant regeneration in a number of species by stimulating production of axillary and/or adventitious shoots or somatic embryos (Huetteman and 2o Preece 1993; Lu 1993; Murthy et al. 1998). In cereals, TDZ-containing media have been used to promote shoot induction from wheat (Hong and Zhen 1999; Shan et al.
2000), barley (Shan et al. 2000) and rice (Tian 1994; Azria and Bhalla 2000) calli.
Gupta and Conger (1998) used a combination of TDZ and 2,4-D in the medium to induce multiple shoots directly from intact seedlings of switchgrass, Panicum virgatum. Nodal explants of Bambusa edulis have also been induced to produce multiple shoots in response to TDZ (Lin and Chang 199$). Similarly for sugarcane, calli induced from immature inflorescences produced shoots on a TDZ-containing medium (Gallo-Meagher et al. 2000).
Studies are constantly being conducted to improve callus production and regeneration from barley by identifying explant systems that are more amenable to in vitro culture and improving media formulations.
SUMMARY OF THE INVENTION
1o This invention relates to the determination of regeneration efficiencies of LB/AM and mature embryos as compared to the regeneration frequency from immature scutella, when treated with TDZ.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1, a,b,c are graphs illustrating the effects of culture media composition on regeneration of barley plants.
Figure 2 a,b are graphs illustrating the effect of TD Z on short induction frequencies in barley LB/AM and mature embryos 2o DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods Seeds of CDC Dawn, CDC McGwire, CDC Copeland and BT459 were obtained from the Crop Development Centre, University of Saskatchewan. CDC
Dawn and CDC McGwire are 2-rowed hulless feed barley varieties, whereas CDC
Copeland and BT459 are 2-rowed and 6-rowed malting barley varieties, respectively.
Seeds were surface-sterilized by shaking in commercial bleach for 45 minutes, rinsed in sterile deionized water and imbibed overnight at 4°C. Mature embryos were subsequently isolated using a dissection microscope. For LB/AM culture, surface-sterilized seeds were germinated for three days in the dark on an agar-solidified MS
(Murashige and Skoog 1962) basal medium. Apical meristems were excised along with the basal portion of the leaf located in the vicinity of the junction between shoot and root. For isolation of scutellar explants, caryopses harvested 10-12 days post-anthesis were de-hulled, surface-sterilized in 5% commercial bleach for five minutes 1o and rinsed in sterile deionized water: The scutella were isolated using a dissection microscope. All isolated explants were incubated on solid media in the dark at 24~ 1 °C for three weeks. Thereafter, the cultures were exposed to low light intensity (100 pmol m 2 s I) for a week and subsequently transferred to fresh media and incubated at 231°C in high light intensity (350 pmol nri 2 s').
Subculture to fresh medium was performed every three weeks for matuxe embryo and LB/AM cultures.
Scutella-derived calli were transferred to a regeneration medium after three to four weeks. Shoots produced from all explants were transferred to an agar-solidified MS
medium containing 1 mglL indolebutyric acid for rooting.
All culture media contained MS salts (Murashige and Skoog 1962), B5 vitamins (Gamborg et al. 1968), 3% maltose, plant growth regulators as listed in Table 1 and 0.8% agar. With the exception of medium MBar (Weir et al.
submitted), all media contained 1.4 g/L enzymatic casein hydrolysate (Sigma Chemical Co., MO, USA), 0.7 glL L-proline and 5 pM copper sulphate.
Seeds were surface-sterilized by shaking in commercial bleach for 45 minutes, rinsed in sterile deionized water and imbibed overnight at 4°C. Mature embryos were subsequently isolated using a dissection microscope. For LB/AM culture, surface-sterilized seeds were germinated for three days in the dark on an agar-solidified MS
(Murashige and Skoog 1962) basal medium. Apical meristems were excised along with the basal portion of the leaf located in the vicinity of the junction between shoot and root. For isolation of scutellar explants, caryopses harvested 10-12 days post-anthesis were de-hulled, surface-sterilized in 5% commercial bleach for five minutes 1o and rinsed in sterile deionized water: The scutella were isolated using a dissection microscope. All isolated explants were incubated on solid media in the dark at 24~ 1 °C for three weeks. Thereafter, the cultures were exposed to low light intensity (100 pmol m 2 s I) for a week and subsequently transferred to fresh media and incubated at 231°C in high light intensity (350 pmol nri 2 s').
Subculture to fresh medium was performed every three weeks for matuxe embryo and LB/AM cultures.
Scutella-derived calli were transferred to a regeneration medium after three to four weeks. Shoots produced from all explants were transferred to an agar-solidified MS
medium containing 1 mglL indolebutyric acid for rooting.
All culture media contained MS salts (Murashige and Skoog 1962), B5 vitamins (Gamborg et al. 1968), 3% maltose, plant growth regulators as listed in Table 1 and 0.8% agar. With the exception of medium MBar (Weir et al.
submitted), all media contained 1.4 g/L enzymatic casein hydrolysate (Sigma Chemical Co., MO, USA), 0.7 glL L-proline and 5 pM copper sulphate.
The number of shoots regenerated from each explant was counted after nine to ten weeks of culture. The general linear model was used for analysis of variance of the data using the SAS software package, Version 8.1~ (SAS Institute Inc., Gary, NC).
Results and Discussion To develop a faster and more reproducible system for barley regeneration, we have tested whether mature embryos and/or LB/AM compare in efficiency with immature scutella for production of shoots. These experiments were conducted with four barley genotypes of potential commercial value for western Canada. The genotypes used show poor tissue culture response as compared to model barley cultivars, but are prime targets for improvement of transformation systems for barley.
Two different culture media, MBar and MB l, were first tested for induction of callus from scutella cultures. MBar has previously been used for culture of barley scutella for callus induction and subsequent regeneration of mature plants (Weir et al.
submitted). Medium MB l differs from MBar by inclusion of enzymatic casein hydrolysate, L-proline and high levels of CuS04; as these factors have been reported to improve barley regeneration (Cho et al. 1998; Dahleen 1995). Comparison of calli formed on the two media showed that the MB 1 medium stimulated production of a more compact, nodular and yellowish embryogenic callus than the MBar medium 2o for all four genotypes (data not shown). Therefore, all subsequent callus induction steps were conducted using the MB 1 medium.
Shoot regeneration from calli produced from scutella and LB/AM cultures on MB 1 medium proceeded for six weeks on four media that differed in 2,4-D and BAP
concentrations (media MB1, MBR1, MBR3 and MBR4 in Table 1). Forregeneration fi from scutella-derived calli, a comparison with the previously used MBar regeneration medium was also earned out {Fig. la). Statistical analysis for the number of shoots produced per scutellum showed that there was a significant genotype x medium interaction (p<0.0001 ). All four genotypes showed the lowest regeneration response on MBar medium and the highest regeneration frequencies on media containing 1.0 (MBR3) or 2.0 mg/L BAP (MBR4) (Fig. l a). MBR_4 was the best medium for CDC
Dawn, CDC McGwire and CDC Copeland, wherea;> BT459 showed about the same response on MBR3 and MBR4. Among the four genotypes, CDC McGwire was most responsive with an average of 2.6 shoots produced per scutellum.
1o The regeneration response from LB/AM-derived calli also showed a significant genotype x medium interaction (p<0.0001 ). For LB/AM calli, the stimulatory effect on shoot induction by high BAP levels seen for scutella was not evident, except for CDC Copeland (Fig. 1b). This genotype responded best on the MBR3 medium, producing on average three shoots per explant. For the other three genotypes, medium MBR4 was slightly better than the other media and induced 1.0 to 1.5 shoots per explant. The LB/AM culture system provided a simple system for callus induction and gave a regeneration response comparable to that from scutella explants. The main advantage of the LB/AM culture is that explants can be obtained within three days of germination of mature seeds and there is no requirement for maintaining a constant supply of donor plants. Excision of LB/AM explants requires use of a dissection microscope, but it is more expeditious than scutella isolation.
As reported by Kapur ( 1991 ), we observed that callus induction from LB/AM
cultures could only be achieved when the apical meristem was present within the excised explant. This observation contradicted a recent study by Pasternak et al.
(2000), who reported callus formation followed by plant regeneration from barley leaf base segments devoid of the apical meristem. The reason for the difference in tissue culture response of LBIAM in these two studies might be that explants used by Pasternak et al. (2000) were from older seedlings (10-20 days) than explants used in our experiments (three days). The effect of the apical meristem and/or age of explant tissue on induction of embryogenic callus tissue in LB/AM explants is not known and will require further studies.
To explore the possibility of using a readily accessible plant material for regeneration, mature embryos were tested as a source of explants. Culture of mature 1o embryos on callus induction medium MB1 resulted in a watery, non-embryogenic callus that appeared similar to the mature embryo-derived type A callus described by Lupotto (1984). As reported by Lupotto (1984), we also found that the type A-like callus could form embryogenic callus upon subculture; although the induction frequency of such embryogenic callus was very low (data not shown).
Since the induction of callus tissue from matrare embryos was insufficient for further regeneration, we tested to see if induction of multiple shoots could be obtained without a callus interphase. In this experiment, excised explants were cultured directly on regeneration media MBRI, MBR3 and MBR4. After nine weeks of culture, 30-60 °lo of the mature embryos of all four genotypes incubated on the different media produced multiple shoots (Fig. lc),. The direct shoot production induced from mature embryos showed a significant genotype x medium interaction (p<0.0001). Medium MBl was least effective and medium MBR4 most effective.
Between 1.5 to 3.5 shoots were obtained per explant with the highest number recorded for CDC Dawn. Overall; direct shoot induction frequencies obtained from mature embryos on medium MBR4 were comparable to those obtained with calli derived from LB/AM and scutella on medium MBR4 incubated for the same length of time (Fig. la, 1b). However, the simplicity and rapid production of shoots from the mature embryo culture strongly favoured its use over the alternative explant sources.
In an attempt to improve plant regeneration for the barley genotypes used in this study, TDZ was included in the medium to evaluate its ability to induce multiple shoots directly as demonstrated in switchgrass by Gupta and Conger (1998). A
combination' of 1.0 mg/L TDZ and 1.0 mg/L 2,4-D in the medium (MBTS) was first tested to simulate the switchgrass experiment. Another medium; MBT6, containing l0 1.0 mg/L 2,4-D and 2 mglL TDZ was also tested. TTeither MBTS nor MBT6 medium increased shoot production from scutella-derived calli. Further increase of the TDZ
levels in the MBTS medium to 3 or 4 mg/L resulted in scutella with a scorched appearance within a few days of culture. Calli derived from scutella or leaf base/apical meristem cultures were also found to be very sensitive to TDZ
exposure, and turned brown and died when incubated on MBTS or MBT6. Similar adverse effects by TDZ have been reported for other plant tissues upon incubation with TDZ
(Lu 1993). Lowering the dose and/or reducing the time of exposure to TDZ can in some cases eliminate negative TDZ effects (Huetteman and Preece 1993). It is possible that hese modifications to the TDZ treatments may have allowed some regeneration from scutella and LB/AM derived calli an this study, but this possibility w$s not explored.
The experiments with LB/AM and mature embryo explants from all four genotypes resulted in production of shoots on the TDZ-containing media MBTS
and MBT6 (Fig.2): Multiple shoots were produced from. LB/AM and mature embryos, without an intervening callus phase, after three to four weeks incubation and increased in number with each subculture. Further. subcultures beyond nine weeks increased the total number of shoots produced per explant to more than 15 depending on the genotype (data not shown), indicating that cultures could possibly be continuously maintained with increased shoot production frequencies. Both and mature embryo shoot production showed a significant genotype x medium interaction (p--0.01). Depending on the genotype, LB/AM produced two to eight shoots per explant (Fig. 2a); whereas mature embryos developed three to eight shoots (Fig. 2b). CDC McGwire showed the best response for both explants. Overall, 1o regeneration frequencies recorded for mature embryos of all four genotypes on the MBTS medium were two-fold higher than the best regeneration frequencies recorded for scutella on medium MBR4 after the same time. Given the simplicity of the mature embryo system, a much improved regeneration system for recalcitrant barley genotypes was achieved through direct multiple shoot induction from mature embryos.
Encouraged by the improved regeneration frequencies obtained for mature embryo explants on media containing 2;4-D and TDZ, we tested the effect of TDZ
as the sole growth regulator on multiple shoot induction. Only mature embryos of genotypes CDC Copeland and BT459 were studied and both produced more shoots on 2o media MBT1 (1.0 mg/L TDZ) and MBT2 (2.0 mg/L), as compared to MBTS
containing both 2,4-D and TDZ. MBTl was a better medium than MBT2 and after nine weeks of incubation induced on average 7.5 and 12 shoots per CDC Copeland and BT459 explant, respectively. Plants regenerated on either media MBTS, MBT6, MBT 1 or MBT2 rooted normally and mature plants did not show any phenotypic aberrations when compared to donor plants.
Regeneration frequencies obtained from mature embryos were close to those reported from immature embryo-derived calli of highly responsive barley cultivars (Wan and Lemaux 1994) that are commonly used for genetic transformation.
Increasing the number of subcultures further increased the total number of shoots produced from the mature embryos, whereas shoot production from callus tissues start to decline after three subcultures, possibly due to loss of regeneration potential of embryogenic calli. We expect that a systematic study of application time and l0 concentration of plant growth regulators in the regeneration media will further improve the barley mature embryo regeneration system presented here to enable its use for genetic transformation.
In this study, we have demonstrated that mature embryos of relatively recalcitrant barley genotypes produce three- to eight-fold more shoots in response to Is TDZ than calli derived from scutella. The use of mature embryos for barley regeneration eliminates the need for immature explant material, and consequently, growth of donor plants. Besides being an expeditious system, the mature embryo system is relatively cost effective in terms of growth chamber and greenhouse space as it has no requirement for resources generally needed for growing donor plants. In 20 addition, the rigorous growth conditions to maintain an optimal physiological state of donor plant material for maximal tissue culture response is eliminated. For these reasons, we anticipate that the mature embryo system .for barley will gain widespread use once genetic transformation has been demonstrated.
References Azria D and Bhalla PL (2000) Plant regeneration from mature embryo-derived callus of Australian rice (Oryza sativa L.) varieties. Aust J Agric Res 51: 305-312 Cheng T-Y and Smith HH (1975) Organogenesis from callus culture of Hordeum vulgare. Planta 123: 307-310 Cho M-J, 3iang W and Lemaux PG (1998) Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism.
Plant Sci 138:229-244 Dahleen LS (1995) Improved plant regeneration from barley callus cultures by 1o increased copper levels. Plant Cell Tiss Org Cult 43: 267-269 Gallo-Meagher M, English &G and Abouzid A (2000) Thidia,zuron stimulates shoot regeneration of sugarcane embryogenic callus. In Vitro Cell Dev Biol-Plant 36: 37-40 Gamborg OLMiller RA and Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151-158 Gupta SD, Conger BV (1998) In vitro differentiation of multiple shoot clumps from intact seedlings of switchgrass. In Vitro Cell I)ev Biol-Plant. 34: 196-202 Hong YX and Zhen Z ( 1999) Improvement in plant regeneration from callus in wheat. Acta Phytophysiologiea Sinica 25(4): 388-394 2o Huetteman CA and Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss Org Cult 33: 10~-119 Kapur R (1991) Studies on the recalcitrance of barlley mesophyll protoplasts with special reference to oxidative stress. Ph D Thesis, Department of Crop Science and Plant Ecology, University of Saskatchewan, Saskatoon; Canada Lin CS and. Chang WC (1998) Micropropagation of Bambusa edudis through nodal explants of field-grown culms and flowering of regenerated plantlets. Plant Cell Rep 17: 617-620 Lu C-Y (1993) The use of thidiazuron in tissue culture. In Vitro Cell Dev Biol 29P:92-96.
Liihrs R and, Lorz H (1987) Plant regeneration in vitro from embryogenic cultures of spring- and winter- type barley varieties. Theor Appl Genet 75:16- 25 Lupotto E (1984) Callus induction and plant regeneration from mature embryos.
Ann Bot 54: 523-529 to Mohanty BD and Ghosh PD (1988) Somatic embryogenesis and plant regeneration from leaf callus ofHordeum vulgare. Ann Bot 61: 551-555 Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1 _'>: 473-497 Murthy BNS, Murch SJ, Saxena PK (1998) Thidiazuron: a potent regulator of in vitro plant morphogenesis. In Vitro Cell Dev Biol-Piant 34: 267-275 Pasternak TP; Rudas VA, Loxz H and Kumlehn J (1999) Embryogenic callus formation and plant regeneration from leaf base segments of barley (Hordeum vulgare L:). J Plant Physiol 155: 371-375 Rengel Z and Jelaska S (1986) Somatic embryogenesis and plant regeneration from seedling tissues of Hordeum vulgate L. J Plant Physiol 124: 385-392 Ritala A, Aspegren K, KurtBn U, Salmenkallio-Marttila M, Mannonen L, Hannus R, Kauppinen V, Teeri TH and Enari T-M (1994) Fertile transgenic barley by particle bombardment of immature embryos. Plant Mol Biol 24:317-25 Saalbach G and Koblitz H (1978) Attempts to initiate callus formation from barley leaves. Plant Sci Lett 13: 165-169 Shun X, Li D and Qu R (2000) Thidiazuron promotes in vitro regeneration of wheat and barley. In Vitro Cell Dev Biol-Plant 36: 207-210 s Tian W ( 1994) Improvement of plant regeneration frequency in vitro in Indica rice.
Acta Genetics. Sinica 21: 215-221 Tingay S, McElroy D, Kalla R, Fieg S, Wang M, 'fhornton S and Brettell R
(1997) Agrobacterium tumefaciens-mediated barley transformation. The Plant J 11:
1o Wan Y and Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104: 37-48 Weigel RC and Hughes KW (1985) Long i:erm regeneration by somatic embryogenesis in barley (Hordeum vulgare L.) tissue cultures derived from apical meristem explants. Plant Cell Tiss Org Cult 5: 151-162 15 Weir BJ, Ganeshan S, Rossnagel BG and Chibbar RN (2001) Optimization of the enhanced regeneration system for selected feed and malting barley genotypes.
Submitted Weir BJ; Ganeshan S, Lai KJ, Caswell K, Rossnagel BG and Chibbar RN (1998) Transforming spring barley using the enhanced regeneration system and 2o microprojectile bombardment. In Vitro Cell Dev Biol 34(3)Part II: 54-A, Abstract # P-1047, March 1998 Zhang S, Williams-Carrier R, Jackson D, Lemaux PG (1998) Expression of CDC2Zm and KNOTTED 1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea Mays L.) and barley (Hordeum vudgare L.). Planta 204: 542-549 Table 1 Concentrations of plant growth regulators in regeneration media.
Plant growth regulator ~mglL) Medium 2,4-D TDZ BAP
MBar 1 - -MBRl 0.05 - 0:1 MBR3 0.05 - 1 MBR4 0.05 - 2 MBT 1 - 1 _
Results and Discussion To develop a faster and more reproducible system for barley regeneration, we have tested whether mature embryos and/or LB/AM compare in efficiency with immature scutella for production of shoots. These experiments were conducted with four barley genotypes of potential commercial value for western Canada. The genotypes used show poor tissue culture response as compared to model barley cultivars, but are prime targets for improvement of transformation systems for barley.
Two different culture media, MBar and MB l, were first tested for induction of callus from scutella cultures. MBar has previously been used for culture of barley scutella for callus induction and subsequent regeneration of mature plants (Weir et al.
submitted). Medium MB l differs from MBar by inclusion of enzymatic casein hydrolysate, L-proline and high levels of CuS04; as these factors have been reported to improve barley regeneration (Cho et al. 1998; Dahleen 1995). Comparison of calli formed on the two media showed that the MB 1 medium stimulated production of a more compact, nodular and yellowish embryogenic callus than the MBar medium 2o for all four genotypes (data not shown). Therefore, all subsequent callus induction steps were conducted using the MB 1 medium.
Shoot regeneration from calli produced from scutella and LB/AM cultures on MB 1 medium proceeded for six weeks on four media that differed in 2,4-D and BAP
concentrations (media MB1, MBR1, MBR3 and MBR4 in Table 1). Forregeneration fi from scutella-derived calli, a comparison with the previously used MBar regeneration medium was also earned out {Fig. la). Statistical analysis for the number of shoots produced per scutellum showed that there was a significant genotype x medium interaction (p<0.0001 ). All four genotypes showed the lowest regeneration response on MBar medium and the highest regeneration frequencies on media containing 1.0 (MBR3) or 2.0 mg/L BAP (MBR4) (Fig. l a). MBR_4 was the best medium for CDC
Dawn, CDC McGwire and CDC Copeland, wherea;> BT459 showed about the same response on MBR3 and MBR4. Among the four genotypes, CDC McGwire was most responsive with an average of 2.6 shoots produced per scutellum.
1o The regeneration response from LB/AM-derived calli also showed a significant genotype x medium interaction (p<0.0001 ). For LB/AM calli, the stimulatory effect on shoot induction by high BAP levels seen for scutella was not evident, except for CDC Copeland (Fig. 1b). This genotype responded best on the MBR3 medium, producing on average three shoots per explant. For the other three genotypes, medium MBR4 was slightly better than the other media and induced 1.0 to 1.5 shoots per explant. The LB/AM culture system provided a simple system for callus induction and gave a regeneration response comparable to that from scutella explants. The main advantage of the LB/AM culture is that explants can be obtained within three days of germination of mature seeds and there is no requirement for maintaining a constant supply of donor plants. Excision of LB/AM explants requires use of a dissection microscope, but it is more expeditious than scutella isolation.
As reported by Kapur ( 1991 ), we observed that callus induction from LB/AM
cultures could only be achieved when the apical meristem was present within the excised explant. This observation contradicted a recent study by Pasternak et al.
(2000), who reported callus formation followed by plant regeneration from barley leaf base segments devoid of the apical meristem. The reason for the difference in tissue culture response of LBIAM in these two studies might be that explants used by Pasternak et al. (2000) were from older seedlings (10-20 days) than explants used in our experiments (three days). The effect of the apical meristem and/or age of explant tissue on induction of embryogenic callus tissue in LB/AM explants is not known and will require further studies.
To explore the possibility of using a readily accessible plant material for regeneration, mature embryos were tested as a source of explants. Culture of mature 1o embryos on callus induction medium MB1 resulted in a watery, non-embryogenic callus that appeared similar to the mature embryo-derived type A callus described by Lupotto (1984). As reported by Lupotto (1984), we also found that the type A-like callus could form embryogenic callus upon subculture; although the induction frequency of such embryogenic callus was very low (data not shown).
Since the induction of callus tissue from matrare embryos was insufficient for further regeneration, we tested to see if induction of multiple shoots could be obtained without a callus interphase. In this experiment, excised explants were cultured directly on regeneration media MBRI, MBR3 and MBR4. After nine weeks of culture, 30-60 °lo of the mature embryos of all four genotypes incubated on the different media produced multiple shoots (Fig. lc),. The direct shoot production induced from mature embryos showed a significant genotype x medium interaction (p<0.0001). Medium MBl was least effective and medium MBR4 most effective.
Between 1.5 to 3.5 shoots were obtained per explant with the highest number recorded for CDC Dawn. Overall; direct shoot induction frequencies obtained from mature embryos on medium MBR4 were comparable to those obtained with calli derived from LB/AM and scutella on medium MBR4 incubated for the same length of time (Fig. la, 1b). However, the simplicity and rapid production of shoots from the mature embryo culture strongly favoured its use over the alternative explant sources.
In an attempt to improve plant regeneration for the barley genotypes used in this study, TDZ was included in the medium to evaluate its ability to induce multiple shoots directly as demonstrated in switchgrass by Gupta and Conger (1998). A
combination' of 1.0 mg/L TDZ and 1.0 mg/L 2,4-D in the medium (MBTS) was first tested to simulate the switchgrass experiment. Another medium; MBT6, containing l0 1.0 mg/L 2,4-D and 2 mglL TDZ was also tested. TTeither MBTS nor MBT6 medium increased shoot production from scutella-derived calli. Further increase of the TDZ
levels in the MBTS medium to 3 or 4 mg/L resulted in scutella with a scorched appearance within a few days of culture. Calli derived from scutella or leaf base/apical meristem cultures were also found to be very sensitive to TDZ
exposure, and turned brown and died when incubated on MBTS or MBT6. Similar adverse effects by TDZ have been reported for other plant tissues upon incubation with TDZ
(Lu 1993). Lowering the dose and/or reducing the time of exposure to TDZ can in some cases eliminate negative TDZ effects (Huetteman and Preece 1993). It is possible that hese modifications to the TDZ treatments may have allowed some regeneration from scutella and LB/AM derived calli an this study, but this possibility w$s not explored.
The experiments with LB/AM and mature embryo explants from all four genotypes resulted in production of shoots on the TDZ-containing media MBTS
and MBT6 (Fig.2): Multiple shoots were produced from. LB/AM and mature embryos, without an intervening callus phase, after three to four weeks incubation and increased in number with each subculture. Further. subcultures beyond nine weeks increased the total number of shoots produced per explant to more than 15 depending on the genotype (data not shown), indicating that cultures could possibly be continuously maintained with increased shoot production frequencies. Both and mature embryo shoot production showed a significant genotype x medium interaction (p--0.01). Depending on the genotype, LB/AM produced two to eight shoots per explant (Fig. 2a); whereas mature embryos developed three to eight shoots (Fig. 2b). CDC McGwire showed the best response for both explants. Overall, 1o regeneration frequencies recorded for mature embryos of all four genotypes on the MBTS medium were two-fold higher than the best regeneration frequencies recorded for scutella on medium MBR4 after the same time. Given the simplicity of the mature embryo system, a much improved regeneration system for recalcitrant barley genotypes was achieved through direct multiple shoot induction from mature embryos.
Encouraged by the improved regeneration frequencies obtained for mature embryo explants on media containing 2;4-D and TDZ, we tested the effect of TDZ
as the sole growth regulator on multiple shoot induction. Only mature embryos of genotypes CDC Copeland and BT459 were studied and both produced more shoots on 2o media MBT1 (1.0 mg/L TDZ) and MBT2 (2.0 mg/L), as compared to MBTS
containing both 2,4-D and TDZ. MBTl was a better medium than MBT2 and after nine weeks of incubation induced on average 7.5 and 12 shoots per CDC Copeland and BT459 explant, respectively. Plants regenerated on either media MBTS, MBT6, MBT 1 or MBT2 rooted normally and mature plants did not show any phenotypic aberrations when compared to donor plants.
Regeneration frequencies obtained from mature embryos were close to those reported from immature embryo-derived calli of highly responsive barley cultivars (Wan and Lemaux 1994) that are commonly used for genetic transformation.
Increasing the number of subcultures further increased the total number of shoots produced from the mature embryos, whereas shoot production from callus tissues start to decline after three subcultures, possibly due to loss of regeneration potential of embryogenic calli. We expect that a systematic study of application time and l0 concentration of plant growth regulators in the regeneration media will further improve the barley mature embryo regeneration system presented here to enable its use for genetic transformation.
In this study, we have demonstrated that mature embryos of relatively recalcitrant barley genotypes produce three- to eight-fold more shoots in response to Is TDZ than calli derived from scutella. The use of mature embryos for barley regeneration eliminates the need for immature explant material, and consequently, growth of donor plants. Besides being an expeditious system, the mature embryo system is relatively cost effective in terms of growth chamber and greenhouse space as it has no requirement for resources generally needed for growing donor plants. In 20 addition, the rigorous growth conditions to maintain an optimal physiological state of donor plant material for maximal tissue culture response is eliminated. For these reasons, we anticipate that the mature embryo system .for barley will gain widespread use once genetic transformation has been demonstrated.
References Azria D and Bhalla PL (2000) Plant regeneration from mature embryo-derived callus of Australian rice (Oryza sativa L.) varieties. Aust J Agric Res 51: 305-312 Cheng T-Y and Smith HH (1975) Organogenesis from callus culture of Hordeum vulgare. Planta 123: 307-310 Cho M-J, 3iang W and Lemaux PG (1998) Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism.
Plant Sci 138:229-244 Dahleen LS (1995) Improved plant regeneration from barley callus cultures by 1o increased copper levels. Plant Cell Tiss Org Cult 43: 267-269 Gallo-Meagher M, English &G and Abouzid A (2000) Thidia,zuron stimulates shoot regeneration of sugarcane embryogenic callus. In Vitro Cell Dev Biol-Plant 36: 37-40 Gamborg OLMiller RA and Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151-158 Gupta SD, Conger BV (1998) In vitro differentiation of multiple shoot clumps from intact seedlings of switchgrass. In Vitro Cell I)ev Biol-Plant. 34: 196-202 Hong YX and Zhen Z ( 1999) Improvement in plant regeneration from callus in wheat. Acta Phytophysiologiea Sinica 25(4): 388-394 2o Huetteman CA and Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss Org Cult 33: 10~-119 Kapur R (1991) Studies on the recalcitrance of barlley mesophyll protoplasts with special reference to oxidative stress. Ph D Thesis, Department of Crop Science and Plant Ecology, University of Saskatchewan, Saskatoon; Canada Lin CS and. Chang WC (1998) Micropropagation of Bambusa edudis through nodal explants of field-grown culms and flowering of regenerated plantlets. Plant Cell Rep 17: 617-620 Lu C-Y (1993) The use of thidiazuron in tissue culture. In Vitro Cell Dev Biol 29P:92-96.
Liihrs R and, Lorz H (1987) Plant regeneration in vitro from embryogenic cultures of spring- and winter- type barley varieties. Theor Appl Genet 75:16- 25 Lupotto E (1984) Callus induction and plant regeneration from mature embryos.
Ann Bot 54: 523-529 to Mohanty BD and Ghosh PD (1988) Somatic embryogenesis and plant regeneration from leaf callus ofHordeum vulgare. Ann Bot 61: 551-555 Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1 _'>: 473-497 Murthy BNS, Murch SJ, Saxena PK (1998) Thidiazuron: a potent regulator of in vitro plant morphogenesis. In Vitro Cell Dev Biol-Piant 34: 267-275 Pasternak TP; Rudas VA, Loxz H and Kumlehn J (1999) Embryogenic callus formation and plant regeneration from leaf base segments of barley (Hordeum vulgare L:). J Plant Physiol 155: 371-375 Rengel Z and Jelaska S (1986) Somatic embryogenesis and plant regeneration from seedling tissues of Hordeum vulgate L. J Plant Physiol 124: 385-392 Ritala A, Aspegren K, KurtBn U, Salmenkallio-Marttila M, Mannonen L, Hannus R, Kauppinen V, Teeri TH and Enari T-M (1994) Fertile transgenic barley by particle bombardment of immature embryos. Plant Mol Biol 24:317-25 Saalbach G and Koblitz H (1978) Attempts to initiate callus formation from barley leaves. Plant Sci Lett 13: 165-169 Shun X, Li D and Qu R (2000) Thidiazuron promotes in vitro regeneration of wheat and barley. In Vitro Cell Dev Biol-Plant 36: 207-210 s Tian W ( 1994) Improvement of plant regeneration frequency in vitro in Indica rice.
Acta Genetics. Sinica 21: 215-221 Tingay S, McElroy D, Kalla R, Fieg S, Wang M, 'fhornton S and Brettell R
(1997) Agrobacterium tumefaciens-mediated barley transformation. The Plant J 11:
1o Wan Y and Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104: 37-48 Weigel RC and Hughes KW (1985) Long i:erm regeneration by somatic embryogenesis in barley (Hordeum vulgare L.) tissue cultures derived from apical meristem explants. Plant Cell Tiss Org Cult 5: 151-162 15 Weir BJ, Ganeshan S, Rossnagel BG and Chibbar RN (2001) Optimization of the enhanced regeneration system for selected feed and malting barley genotypes.
Submitted Weir BJ; Ganeshan S, Lai KJ, Caswell K, Rossnagel BG and Chibbar RN (1998) Transforming spring barley using the enhanced regeneration system and 2o microprojectile bombardment. In Vitro Cell Dev Biol 34(3)Part II: 54-A, Abstract # P-1047, March 1998 Zhang S, Williams-Carrier R, Jackson D, Lemaux PG (1998) Expression of CDC2Zm and KNOTTED 1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea Mays L.) and barley (Hordeum vudgare L.). Planta 204: 542-549 Table 1 Concentrations of plant growth regulators in regeneration media.
Plant growth regulator ~mglL) Medium 2,4-D TDZ BAP
MBar 1 - -MBRl 0.05 - 0:1 MBR3 0.05 - 1 MBR4 0.05 - 2 MBT 1 - 1 _
Claims
1. The invention as substantially described herein.
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| CN102599059A (en) * | 2012-03-28 | 2012-07-25 | 中国农业科学院作物科学研究所 | Method for improving tissue culture regeneration rate of wheat genotype immature embryo with low regeneration capacity |
| CN103947550A (en) * | 2014-04-20 | 2014-07-30 | 浙江省农业科学院 | Tissue culture method for directly growing seedling from barley embryo and culture medium used in method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102599059A (en) * | 2012-03-28 | 2012-07-25 | 中国农业科学院作物科学研究所 | Method for improving tissue culture regeneration rate of wheat genotype immature embryo with low regeneration capacity |
| CN103947550A (en) * | 2014-04-20 | 2014-07-30 | 浙江省农业科学院 | Tissue culture method for directly growing seedling from barley embryo and culture medium used in method |
| CN103947550B (en) * | 2014-04-20 | 2016-04-13 | 浙江省农业科学院 | The direct seedling tissue culture method of barley immature embryos and used medium |
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