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Donor-Plant Environment Effects on Regeneration from Barley Embryo-Derived Callus.

Barley transformation techniques use tissue culture systems that require high rates of green plant regeneration for acceptable transformation efficiencies. Regeneration from embryo-derived callus is controlled by several genes (Komatsuda et al., 1989; Mano et al., 1996; P. Bregitzer, 1998, personal communication), and there is a large range of responses from different genotypes (Baillie et al., 1993; Borod'ko et al., 1991; Bregitzer, 1992) In the USA, most transformation experiments have used the cultivar Golden Promise because it consistently regenerates sufficient numbers of green plants. U.S. malting cultivars respond poorly to standard tissue culture methods, a significant obstacle to their use in transformation.

Research to improve regeneration in North American malting cultivars has concentrated on media composition and growth regulator regimes (Bregitzer et al., 1995, 1998; Dahleen, 1995). During these experiments, it was noted that while high levels of regeneration were possible from greenhouse-grown donor plants, levels varied greatly from experiment to experiment. Some of this variation could be attributed to controllable effects, such as chemical damage or insufficient watering (P. Bregitzer, 1998, personal communication). Other variation appeared to be seasonal, perhaps caused by differences in light levels and temperature. Regeneration from donor plants grown in controlled growth chamber conditions in these experiments was less dependent on planting time. Because growth-chamber space can be limited, the greenhouse often is used for growing donor plants for tissue culture and transformation.

The objective of this research was to evaluate regeneration from barley tissue culture of donor plants grown under growth-chamber and greenhouse conditions, planted on four different dates, to determine optimal greenhouse planting dates.


Plant Material

Single plant-derived lines of the barley cultivars Morex and Golden Promise were planted in the growth chamber and greenhouse on four dates: 1 June 1996, 1 Sept. 1996, 1 Dec. 1996, and 1 Mar. 1997. Single seed were placed in 15-cm clay pots in a potting mix (Sunshine mix #1, SunGro Horticulture Canada Ltd., Bellevue, WA) supplemented with a slow-release fertilizer (Osmacote 14-14-14, Scotts-Sierra Horticultural Products Co., Marysville, OH).(1) Two weeks after planting, plants were treated with the systemic insecticide, Marathon (Olympic Horticultural Products Co., Mainland, PA). The growth chamber was maintained at 20 [degrees] C during the day) and 16 [degrees] C at night, with 16 h of light from a mixture of fluorescent and incandescent bulbs. Natural lighting in the greenhouse was supplemented with sodium vapor bulbs to provide 16 h of light per day. The sodium vapor lamps provided 90 W [m.sup.-2] irradiance at bench level up to 144 W [m.sup.-2] at canopy height ([approximately equals] 80 cm). Full sunlight can provide from 360 W [m.sup.-2] irradiance at bench level to 450 W [m-.sup.2] at canopy height. Greenhouse temperatures were monitored using a thermograph and average daily solar radiation readings were obtained from the North Dakota Agricultural Weather Network.

Tissue Culture

Approximately 2 wk after anthesis, caryopses with embryos 2 to 3 mm long were collected. Time from planting to culture ranged from 49 to 138 d (Table 1). Seed were surface sterilized in 70% ethanol for 1 min, 50% commercial bleach (2.625% Na hypochlorite) for 5 min, followed by three 5-min rinses in sterile water. Immature embryos were excised from seed under sterile conditions and cut longitudinally through the root-shoot axis. Bisected embryos were placed on modified MS (Murashige and Skoog, 1962) inorganic salts, with 1.25 mg [L.sup.-1] Cu[SO.sub.4], 1 g [L.sup.-1] casein hydrolysate, 0.25 g [L.sup.-1] myo-inositol, 30 g [L.sup.-1] maltose, 4.5 g [L.sup.-1] 2,4 dichlorophenoxyacetic acid (2,4-D), and solidified with 3.5 g [L.sup.-1] Phytagel (Sigma, St. Louis, MO). Media components were autoclaved in three parts (maltose; [KH.sub.2][PO.sub.4], EDTA, and [FeSO.sup.4]; remainder of components) to prevent browning (Bregitzer et al., 1998), and combined before pouring 50 mL into each 25 by 100 mm petri plate.

Table 1. Individual effects of genotype, donor plant growth environment, and planting date on green plant regeneration in barley.
                                            Planting date

                                        June 1996        September

                                    Green      Days to     Green
Genotype         Environment        plants     culture     plants

Morex            Growth chamber     81.7 de     70-79      71.7 ef
Morex            Greenhouse         69.1 ef     49-50      51.9 fg
Golden Promise   Growth chamber    114.3 bc      105      101.0 cd
Golden Promise   Greenhouse         96.4 cd     77-84     115.9 bc

                                    Planting date

                  September     December 1996         March 1997

                   Days to    Green     Days to    Green    Days to
Genotype           culture    plants    culture    plants   culture

Morex              91-114     79.4 de      78       86.8 de    86
Morex                62       24.5 h       50       52.0 fg    56
Golden Promise      138       81.0 de     105      152.9 a    112
Golden Promise     77-84      42.5 gh      49      127.4 b     58

([dagger]) Mean number of green plants regenerated per petri plate. Means for green plants/plate across all planting dates followed by the same letter are not significantly different (P < 0.01).

Four embryos were placed on each plate, with 25 plates for each cultivar-environment-date combination. Cultures were incubated in the dark at 23 to 25 [degrees] C for 4 wk. At that time, half of the callus from the three most vigorously growing embryos on each plate was transferred to fresh medium lacking 2,4-D but containing 2.5 mg [L.sup.-1] 6-benzylaminopurine (BAP) for 1 wk. Calli were then placed on regeneration medium lacking growth regulators under fluorescent lights at 21 to 23[degrees]C to promote green plant development. Four weeks later, albino and vigorously growing green plants were counted. The remaining small shoots and green calli were transferred to half-strength regeneration medium containing 30 g [L.sup.-1] sucrose instead of maltose, in 75 by 65 by 65 mm culture boxes. Final albino and vigorous green plant counts were taken 4 wk later. First and second counts were summed to give total green and albino plant regeneration.

Data Analysis

Analysis of variance (ANOVA) and mean separation were conducted using CoStat computer software (CoHort Software, Minneapolis, MN). Data were analyzed as a 2 by 2 by 4 random effects factorial design with cultivar, growth environment, and planting date as main effects. Experimental units were the total regeneration from individual plates. The error mean square from the ANOVA was used to calculate the LSD for each comparison. Numbers of green and albino regenerants were compared across genotype, donor-plant growth environment, and planting date.


These experiments were designed because of some observations made on a set of experiments conducted for other reasons in 1994 and 1995. Comparison of regeneration rates from explant donor plants grown in the greenhouse indicated two- to eightfold differences in regeneration depending on the planting date. Controlled environmental conditions in the growth chamber are recommended for barley transformation procedures because of high and consistent plant regeneration rates (Lemaux et al., 1995), but growth-chamber space can be limited. The best regeneration rates from greenhouse-grown plants appeared to be as good or better than the rates from growth chamber-grown plants. A more complete understanding of the relationship between donor-plant growth environment and regeneration response is needed to take advantage of greenhouse space when growth-chamber space is limited.

Growth Environment

Conditions in the growth chamber were relatively constant, with only small changes in light levels as bulbs burned out and were replaced. Greenhouse conditions varied in light levels and temperature (Table 2). Average solar radiation varied greatly from month to month, as expected in a northern climate. Daylight hours on the fifteenth of each month varied from approximately 8.7 h in December to 15.7 h in June. The sodium vapor lamps that were the sole source of light for up to 7 h per day during the winter months only provided 25 to 32% of the irradiance to the growing plants as full sunlight, which can result in reduced plant vigor and fertility. The average greenhouse temperatures did not vary greatly, ranging from 21 to 26 [degrees] C. High temperature spikes were common during months with higher solar radiation, although they usually lasted less than 1 h. Temperature drops were less frequent and less extreme.
Table 2. Environmental factors affecting plant growth conditions
in the greenhouse from June 1996 through May 1997.

             Average daily     Greenhouse temperature
            solar radiation
Month       (KJ m)([dagger])   Daily average Minimum   Maximum
June             13.22         23-26            18        40
July             12.95         24-26            19        34
August           12.26         23-26            19        32
September         8.15         23-26            17        32
October           5.26         21-23            12        29
November          3.25         22-24            19        27
December          2.39         24-26            14        29
January           3.18         23-26            17        27
February          5.19         23-26            20        29
March             6.33         23-26            20        31
April            10.97         22-24            19        36
May              11.85         21-23            18        34

([dagger]) North Dakota Agricultural Weather Network.

Days to culture was much shorter for greenhouse-grown plants than for growth chamber-grown plants. This was probably due to the cooler temperatures in the growth chamber, 20 [degrees] C during the day and 16 [degrees] C at night. These lower temperatures typically slow overall plant growth and delayed heading in both cultivars.

Plant Regeneration

Analysis of variance indicated that the effects of genotype, planting date, and donor-plant growth environment on green plant regeneration were highly significant (P [is less than] 0.001). Environment had no impact on albino frequency, although the effects of planting date (P [is less than] 0.05) and genotype (P [is less than] 0.001) were significant. The two-way interactions between date and both environment and genotype (P [is less than] 0.001), and between environment and genotype (P [is less than] 0.05) were significant for green plant regeneration. Only the interactions between date and environment (P [is less than] 0.001) and date and genotype (P [is less than] 0.05) were significant for albino plant regeneration. The three-way interaction was not significant for either trait.

As expected, the Midwestern U.S. six-rowed malting barley cultivar Morex regenerated significantly fewer green plants and more albino plants than Golden Promise (Table 3). Golden Promise has been used as a model cultivar for transformation because of its high regeneration rates. Donor-plant growth environment also affected regeneration, as growth chamber-grown plants regenerated significantly more green plants. Donor-plant environment did not affect albino frequency.

Table 3. Mean effects of genotype, donor plant growth environment, and planting date on green and albino plant regeneration in barley.([dagger])
Variable                Green        Albino

  Morex                  64.7 b      6.1 a
  Golden Promise        104.2 a      1.1 b
  Growth chamber         96.3 a      1.1 a
  Greenhouse             72.2 b      1.3 a
Date of planting
  June 1996              90.3 b      0.9 b
  September 1596         85.3 b      1.3 ab
  December 1996          57.2 c      1.5 a
  March 1997            105.3 a      1.1 ab

([dagger]) Mean number of green and albino plants regenerated per petri plate. Means within a variable followed by different letters are significantly different at P < 0.01.

Date of planting had significant effects on both albino and green plant regeneration. The only differences in albino plant regeneration within cultivars at one planting date was for December 1996. For both Morex and Golden Promise planted on this date, cultures derived from greenhouse-grown plants regenerated significantly more albinos than cultures derived from growth chamber-grown plants (data not shown).

The effects of planting date on green plant regeneration from the two cultivars were similar (Table 1). The cultures of embryos from seeds sown in December resulted in significantly fewer green regenerants per plate. Only small differences were seen between other greenhouse planting dates. The best greenhouse regeneration rate for Morex, 69.1 green plants per plate, was not significantly different than the regeneration rate from growth chamber-grown Morex donor plants (Table 1). Growth chamber-grown Morex regenerated consistently high numbers of green plants all year. Regeneration from Golden Promise grown in the growth chamber was surprisingly variable, with almost a twofold difference between the December and March planting date responses.

Greenhouse environmental effects on Morex regeneration closely followed the change in average solar radiation (Tables 1 and 2). Temperature spikes had little effect, probably because Morex was bred for the upper Midwestern USA, where summer temperatures of 30 to 36 [degrees] C are common. The 40 [degrees] C temperature spike in June occurred 2 to 3 wk after emergence, before much development of floral structures. Golden Promise also showed a decrease in regeneration from embryos grown under low average solar radiation (Tables 1 and 2), and appears sensitive to the frequent high temperature spikes during June and July. We have observed similar decreases in Morex regeneration from plants grown in the summer when temperature spikes were more frequent and of longer duration.


Donor-plant growth environment has been extensively studied for haploid production from anthers and microspores (Pickering and Devaux, 1992), with most emphasis on temperature. Yet reviews of factors affecting regeneration from callus cultures derived from embryos rarely mention donor plant growth environment (Dunwell, 1986; Mohan Jain et al., 1988; Jahne-Gartner and Lorz, 1996). Only a few studies describe donor plant growth conditions (Baillie et al., 1993; Bregitzer, 1992; Dahleen, 1995; Goldstein and Kronstad, 1986), but those that use the greenhouse do not state in which months the plants were grown. Results from this experiment show that planting date can significantly affect regeneration response from greenhouse-grown donor plants.

Sufficient solar radiation is present in the greenhouse year round to promote flowering in barley. The fivefold difference in average daily solar radiation between June and December affected seed set in the greenhouse. Plants started in December 1996 had lower seed set and some completely sterile spikes. The reduced fertility indicates that the lower light levels in December had effects on plant vigor. Embryos cultured from these plants were similar in size to those from plants grown at other dates. The reduced solar radiation in December may have slowed embryo development; so those embryos would be older than the same-sized embryos from plants started in March through September. Hess and Carman (1998) suggest that wheat (Triticum aestivum L.) embryos lose regeneration capacity with age, as hormone levels change in ovulo. Their data indicate that temperature can alter the timing of these hormone changes. It is possible that the rate of embryo development and the in ovule hormone levels also are influenced by the amount of solar radiation received by the donor plants, and these factors may affect regeneration rates in barley. Greenhouse temperature differences during the year of these tests were minimal and had little impact on donor-plant growth and green plant regeneration. In previous years when greenhouse temperatures in June and July were higher, similar reductions in donor-plant fertility and green plant regeneration to those seen in December 1996 were observed.

Data from this study shows that the impact of donorplant growth environment on regeneration can be as large as the effects of genotype and media composition. Overall, green plant regeneration was higher and less variable from growth chamber-grown plants. These consistently high rates are needed for efficient barley transformation. Sufficient regeneration from greenhousegrown donor plants can be reached when natural light levels are high and temperatures are moderate.

Abbreviations: ANOVA, analysis of variance; BAP, 6-benzylaminopurine 2,4-D, 2,4 dichlorophenoxyacetic acid.


The author thanks Mrs. Luming Brewer for culture maintenance, Mr. Bill Morgan for maintaining plants in the greenhouse, and Ms. Rachel Lone for media preparation.

(1) Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable. Lynn S. Dahleen(*)


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Lynn S. Dahleen(*)

Cereal Crops Research Unit, USDA-ARS, Northern Crop Science Laboratory, P.O. Box 5677 SU Station, Fargo, ND 58105. Received 29 June 1998. (*) Corresponding author (
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Author:Dahleen, Lynn S.
Publication:Crop Science
Date:May 1, 1999
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