4
Annals of Botany 80 : 561–564, 1997 SHORT COMMUNICATION A Seed Treatment for Eliminating Non-hybrid Plants when using Atrazine Resistance as a Genetic Marker for Hybrid Seed Production A. J. CONNER and M. C. CHRISTEY New Zealand Institute for Crop & Food Research Ltd, Priate Bag 4704, Christchurch, New Zealand Received : 23 December 1996 Accepted : 12 May 1977 Brassica plants, with a combination of chloroplasts encoding triazine resistance and mitochondria encoding cytoplasmic male sterility, offer new opportunities for hybrid seed production. Such plants can be grown as a random mixture with a male parent, thereby allowing more effective pollen transfer for greater efficiency of hybrid seed production. Harvested seed consists of hybrid seed from the ‘ female ’ parent, which is triazine-resistant, and non- hybrid, triazine-sensitive seed resulting from self pollination of the ‘ male ’ parent. This study demonstrates that triazine-sensitive broccoli (Brassica oleracea L. var. italica) progeny can easily be eliminated by allowing the seed to imbibe a solution of 10 g l -" atrazine overnight then drying-back the seed for subsequent germination. Such a treatment prevents triazine-sensitive broccoli from developing beyond the cotyledon stage following germination, and remains effective for at least 35 years after seed treatment. The growth of triazine- resistant broccoli plants is unaffected by this seed treatment. # 1997 Annals of Botany Company Key words : Atrazine, Brassica oleracea, broccoli, hybrid seed, triazine resistance, seed treatment. INTRODUCTION Recent advances in plant cellular and molecular biology have greatly simplified the development of herbicide resistance in a wide range of crops. The ease with which these genetic manipulations can be made has opened up a range of new applications for herbicide resistance in crop protection, agronomic practice and seed production (Conner and Field, 1995). One approach for the transfer of herbicide resistance in crops involves the somatic hybridization of isolated protoplasts. The regeneration of plants following protoplast fusion can allow the artificial synthesis of cybrids, cytoplasmic hybrids with new combinations of organelle genomes (mitochondria and chloroplasts). Such plants cannot be developed through conventional sexual hybridi- zation due to the uniparental, maternal inheritance of organelle genomes (Conner and Meredith, 1989). The development of cybrid brassica plants with a combination of chloroplasts encoding triazine resistance and mitochondria encoding cytoplasmic male sterility has been successful in canola (Yarrow et al., 1986 ; Barsby et al., 1987 ; Chuong et al., 1988; Kao et al., 1991) and broccoli (Christey, Makaroff and Earle, 1991). These plants have important applications for hybrid seed production where inadequate pollen transfer is a common problem, resulting in poor yield of hybrid seed on the ‘female’ parent. The transfer of pollen from the ‘male’ parent to the ‘ female ’ parent can be substantially improved if the distance between the two parents can be reduced. If the ‘female’ parent has a genetic marker for herbicide resistance, the ‘male’ and ‘female’ parents could be grown as a random mixture, rather than as separate alternating blocks in the field, resulting in more efficient pollen transfer, and therefore higher yield and more economical production of hybrid seed (Beversdorf, Erickson and Grant, 1985 ; Conner and Field, 1995). The seed harvested from this mixed population will consist of hybrid seed from the ‘female’ parent, which is herbicide-resistant, and non-hybrid seed resulting from self pollination of the ‘ male ’ parent, which will be herbicide- sensitive. The non-hybrid seed can easily be eliminated by applying the appropriate herbicide. An environmental concern of this approach to hybrid seed production is the dependence upon field applications of atrazine to eliminate the non-hybrid progeny. To circumvent this problem, this paper presents a simple seed treatment that effectively eliminates the atrazine-sensitive brassica progeny. We demonstrate that this approach remains effective during seed storage for at least 35 years, with no effect on the growth of atrazine-resistant progeny. MATERIALS AND METHODS Plant material Two atrazine-resistant, cytoplasmic male sterile-nigra broc- coli (Brassica oleracea L. var. italica) lines, 16A and 16D, with a nuclear background of Green Comet (Christey et al., 1991 ; Christey and Earle, 1994), were crossed as female parents to the atrazine-sensitive, male-fertile broccoli culti- var Green Comet (Harris Moran Seed Co). This generated the populations 16AGC and 16DGC, which were compared to Green Comet (GC) in this study. 0305-7364}97}10056104 $25.00}0 bo970464 # 1997 Annals of Botany Company

A Seed Treatment for Eliminating Non-hybrid Plants when using Atrazine Resistance as a Genetic Marker for Hybrid Seed Production

Embed Size (px)

Citation preview

Page 1: A Seed Treatment for Eliminating Non-hybrid Plants when using Atrazine Resistance as a Genetic Marker for Hybrid Seed Production

Annals of Botany 80 : 561–564, 1997

SHORT COMMUNICATION

A Seed Treatment for Eliminating Non-hybrid Plants when using Atrazine

Resistance as a Genetic Marker for Hybrid Seed Production

A. J. CONNER and M. C. CHRISTEY

New Zealand Institute for Crop & Food Research Ltd, Pri�ate Bag 4704, Christchurch, New Zealand

Received: 23 December 1996 Accepted: 12 May 1977

Brassica plants, with a combination of chloroplasts encoding triazine resistance and mitochondria encodingcytoplasmic male sterility, offer new opportunities for hybrid seed production. Such plants can be grown as a randommixture with a male parent, thereby allowing more effective pollen transfer for greater efficiency of hybrid seedproduction. Harvested seed consists of hybrid seed from the ‘female’ parent, which is triazine-resistant, and non-hybrid, triazine-sensitive seed resulting from self pollination of the ‘male ’ parent.

This study demonstrates that triazine-sensitive broccoli (Brassica oleracea L. var. italica) progeny can easily beeliminated by allowing the seed to imbibe a solution of 10 g l−" atrazine overnight then drying-back the seed forsubsequent germination. Such a treatment prevents triazine-sensitive broccoli from developing beyond the cotyledonstage following germination, and remains effective for at least 3±5 years after seed treatment. The growth of triazine-resistant broccoli plants is unaffected by this seed treatment. # 1997 Annals of Botany Company

Key words : Atrazine, Brassica oleracea, broccoli, hybrid seed, triazine resistance, seed treatment.

INTRODUCTION

Recent advances in plant cellular and molecular biologyhave greatly simplified the development of herbicideresistance in a wide range of crops. The ease with whichthese genetic manipulations can be made has opened up arange of new applications for herbicide resistance in cropprotection, agronomic practice and seed production (Connerand Field, 1995). One approach for the transfer of herbicideresistance in crops involves the somatic hybridization ofisolated protoplasts. The regeneration of plants followingprotoplast fusion can allow the artificial synthesis of cybrids,cytoplasmic hybrids with new combinations of organellegenomes (mitochondria and chloroplasts). Such plantscannot be developed through conventional sexual hybridi-zation due to the uniparental, maternal inheritance oforganelle genomes (Conner and Meredith, 1989).

The development of cybrid brassica plants with acombination of chloroplasts encoding triazine resistanceand mitochondria encoding cytoplasmic male sterility hasbeen successful in canola (Yarrow et al., 1986; Barsby et al.,1987; Chuong et al., 1988; Kao et al., 1991) and broccoli(Christey, Makaroff and Earle, 1991). These plants haveimportant applications for hybrid seed production whereinadequate pollen transfer is a common problem, resultingin poor yield of hybrid seed on the ‘female’ parent.

The transfer of pollen from the ‘male ’ parent to the‘female’ parent can be substantially improved if the distancebetween the two parents can be reduced. If the ‘female’parent has a genetic marker for herbicide resistance, the‘male ’ and ‘female’ parents could be grown as a random

mixture, rather than as separate alternating blocks in thefield, resulting in more efficient pollen transfer, and thereforehigher yield and more economical production of hybrid seed(Beversdorf, Erickson and Grant, 1985; Conner and Field,1995). The seed harvested from this mixed population willconsist of hybrid seed from the ‘female’ parent, which isherbicide-resistant, and non-hybrid seed resulting from selfpollination of the ‘male ’ parent, which will be herbicide-sensitive. The non-hybrid seed can easily be eliminated byapplying the appropriate herbicide.

An environmental concern of this approach to hybridseed production is the dependence upon field applications ofatrazine to eliminate the non-hybrid progeny. To circumventthis problem, this paper presents a simple seed treatmentthat effectively eliminates the atrazine-sensitive brassicaprogeny. We demonstrate that this approach remainseffective during seed storage for at least 3±5 years, with noeffect on the growth of atrazine-resistant progeny.

MATERIALS AND METHODS

Plant material

Two atrazine-resistant, cytoplasmic male sterile-nigra broc-coli (Brassica oleracea L. var. italica) lines, 16A and 16D,with a nuclear background of Green Comet (Christey et al.,1991; Christey and Earle, 1994), were crossed as femaleparents to the atrazine-sensitive, male-fertile broccoli culti-var Green Comet (Harris Moran Seed Co). This generatedthe populations 16A¬GC and 16D¬GC, which werecompared to Green Comet (GC) in this study.

0305-7364}97}100561­04 $25.00}0 bo970464 # 1997 Annals of Botany Company

Page 2: A Seed Treatment for Eliminating Non-hybrid Plants when using Atrazine Resistance as a Genetic Marker for Hybrid Seed Production

562 Conner and Christey—Seed Treatment to Eliminate Atrazine-sensiti�e Plants

Seed treatments

Seed of 16A¬GC, 16D¬GC and Green Comet were leftovernight (18 h) to imbibe a solution of water or 10 g l−"

atrazine, then dried to ambient room conditions, and storedat room temperature for up to 3±5 years. In all seedtreatments there was approx. 10 ml liquid per 100 seeds.

Germination and plant growth

At ten intervals over 3±5 years following seed treatments,seeds were sown at a depth of about 1 cm in a soil mixconsisting of three parts shredded bark (Pinus radiata) : twoparts sand (washed crusher dust), supplemented with 6±4 kgm−$ dolomite lime, 3±32 kg m−$ ‘Osmocote 14–16±1–11±6’(Sierra), 1±4 kg m−$ superphosphate, 120 g m−$ calciumnitrate, 56 g m−$ ferrous source (‘Fetrilon-Combi1’ ; BASF),and 1 kg m−$ trace elements (‘Micromax’ ; Sierra). Seedswere germinated and plants allowed to establish in aglasshouse heated at temperatures below 18 °C and venti-lated above 22 °C. All plants were watered as required.

Seedlings germinating from seed stored for 2±5 and 26months were individually transplanted into small plasticpots (6±5 cm−$) containing the above soil mix at the two tothree true leaf stage.

Data recording and statistical analysis

Seed germination 10 d after sowing and plant estab-lishment beyond the two to three true-leaf stage (30 d aftersowing) were recorded. For plants grown beyond the two tothree true leaf stage, the number of nodes, plant height andshoot weight were determined 4–6 weeks after trans-plantation. Plant growth data were subjected to analysis ofvariance (Sokal and Rohlf, 1969).

RESULTS

Seed germination

During the first 2±5 years of storage after seed treatment,the germination frequency in all three lines of broccoli wasgenerally between 92 and 96%, irrespective of whetherseeds had been soaked in water or 10 g l−" atrazine. After3±5 years the germination frequency had fallen to 78%, butwith no effect of either seed line or treatment. The imbibitionby broccoli seeds of 10 g l−" atrazine clearly had no effect onthe frequency of germination for either triazine-resistant ortriazine-sensitive seeds (Fig. 1A). However, this atrazinetreatment had a marked effect on the appearance of thecotyledons from the atrazine-sensitive Green Comet seeds.Upon germination, these seedlings had slightly bleachedcotyledons (Fig. 1A) with enhanced anthocyanin pigmen-tation, and rapidly became more chlorotic with furthergrowth.

Seedling establishment

The Green Comet seedlings germinating from the atrazineseed treatment quickly deteriorated following germinationand died (Fig. 1B). This effect was apparent followingstorage of seeds for at least 18 months after the atrazine

F. 1. The germination and establishment of broccoli seedlingsfollowing imbibition by seed of 10 g l−" atrazine. A, 7 d after sowing;B, 17 d after sowing. Each photograph includes the triazine-resistantlines 16A¬GC (left) and 16D¬GC (right), and the triazine-sensitive

Green Comet (centre).

42

100

Length of seed storage (months)

See

dlin

g es

tabl

ish

men

t (%

)

12

75

50

25

1 2 4 5 18 24 30 360 3

F. 2. The establishment of broccoli seedlings to the two–three trueleaf stage at 30 d after sowing dried seed following imbibition in wateror 10 g l−" atrazine. E, Triazine-resistant lines 16A¬GC and16D¬GC imbibed in water or atrazine, and triazine-sensitive GreenComet imbibed in water ; +, triazine-sensitive Green Comet imbibed in

atrazine.

treatment (Fig. 2). Longer storage periods (26–42 months)resulted in the establishment of some Green Comet seedlingsbeyond the cotyledon stage, although the majority of these

Page 3: A Seed Treatment for Eliminating Non-hybrid Plants when using Atrazine Resistance as a Genetic Marker for Hybrid Seed Production

Conner and Christey—Seed Treatment to Eliminate Atrazine-sensiti�e Plants 563

8

0

Nu

mbe

r of

nod

es

54

2

GC 16DxGC16AxGC

76

3

1

20

0

Sh

oot

hei

ght

(cm

)

10

5

GC 16DxGC16AxGC

15

25

0

Sh

oot

wei

ght

(g)

5

GC 16DxGC16AxGC

20

15

10

F. 3. The growth of broccoli plants from seeds stored for 26 monthsafter imbibition in water (+) and 10 g l−" atrazine (*) for triazine-sensitive Green Comet (GC) and triazine-resistant lines 16A¬GC and16D¬GC. Each bar represents the mean of 10 replicates. For all plantgrowth measurements, analysis of variance established no significantdifference between all three plant lines whose seeds were imbibed inwater, or 16A¬GC and 16D¬GC imbibed in atrazine (P" 0±05). Thegrowth of Green Comet plants after imbibing seeds in atrazine wassignificantly less than all other treatments (P! 0±001). LSD

!±!&

¯ 0±5,2±8 and 4±0 for number of nodes, shoot height and shoot weight,

respectively.

seedlings were only just alive and did not develop beyondthe two–three true leaf stage (Fig. 2). The few remainingseedlings (! 10%) remained severely stunted, with multiplelateral shoots and small, thickened stems and leaves. Incontrast, there was no apparent difference in the rate ofseedling establishment between broccoli lines 16A¬GC and16D¬GC (ex water or atrazine seed treatments), or GreenComet (ex water seed treatment).

Plant growth

After 2±5 months storage following seed treatment,randomly selected seedlings from broccoli lines 16A¬GCand 16D¬GC (ex water or atrazine seed treatments), andGreen Comet (ex water seed treatment) were individuallytransplanted into pots and grown for an additional 4 weeks.Analysis of variance on the number of nodes (overall mean¯ 6±6), shoot height (overall mean¯ 8±6 cm) and shoot

weight (overall mean¯ 9±9 g) established no significantdifference in growth of the plants from these five treatments(P" 0±05).

When this experiment was repeated 26 months after seedtreatments, the healthiest surviving seedlings from theatrazine-treated Green Comet seeds were also included (Fig.3). As expected, the growth of these atrazine-sensitiveseedlings was substantially less (P! 0±001) than the otherfive treatments, which did not differ from one another.

DISCUSSION

A common problem during F"

hybrid seed production isinadequate pollen transfer, resulting in poor yield of hybridseed by the ‘female’ parent. Herbicide resistance genes canbe used as genetic markers to overcome this problem(Conner and Field, 1995). By sowing a male-sterile parentwith herbicide resistance as a random mixture with a pollenparent that is sensitive to the same herbicide, the hybrid seedcan be distinguished by the resistance to the herbicide. Evenwhen the pollen parent comprises only 2–10% of therandom mixture, the closer proximity of the two parents isexpected to result in more efficient pollen transfer, therebyresulting in higher yields of hybrid seed.

The development of cybrid brassica plants with thecombination of chloroplast-encoded triazine resistance andmitochondrial-encoded cytoplasmic male sterility (Yarrowet al., 1986; Barsby et al., 1987; Chuong et al., 1988;Christey et al., 1991; Kao et al., 1991) provides ideal linesfor ‘ female’ parents of F

"hybrid seed. When randomly

planted with a small proportion of a triazine-sensitivepollen donor, it has been proposed that the non-hybrid seedresulting from the self pollination of the pollen parent couldbe prevented from developing by spraying out the pollenparent after flowering, but before seed set or seed harvest(Beversdorf et al., 1985). However, the complete eliminationof such seeds cannot be guaranteed due to difficulties inattaining uniform herbicide application over all individualsin a population, and some seed may still develop on dyingplants. Alternatively, the non-hybrid progeny can beeliminated by applying the herbicide to the next generationof F

"hybrid crops.

In this paper we have demonstrated a simple seedtreatment for the effective elimination of triazine-sensitivebrassica progeny. Germinating seedlings were preventedfrom developing beyond the cotyledon stage after imbibingseeds in a solution of 10 g l−" atrazine. This approachremains effective during seed storage for at least 3±5 yearsafter seed treatment (Fig. 2). The continued growth of thetriazine-resistant lines was unaffected by the seed treatmentwith atrazine (Fig. 3).

This approach will require an oversowing in seed densityto account for those progeny that will be subsequentlyeliminated. Determination of the proportion of non-hybrid,triazine-sensitive seed prior to seed marketing will allowseed volume and sowing rates to be adjusted to account foranticipated losses due to herbicide sensitivity.

A few triazine-sensitive Green Comet plants were ob-served to survive this selection method following seedstorage for more than 2 years after seed treatment (Figs 2

Page 4: A Seed Treatment for Eliminating Non-hybrid Plants when using Atrazine Resistance as a Genetic Marker for Hybrid Seed Production

564 Conner and Christey—Seed Treatment to Eliminate Atrazine-sensiti�e Plants

and 3). However, the growth of these seedlings was severelycompromised, with the majority not developing beyond thetwo–three true leaf stage. The few plants that did reach thisstage of development had very poor vegetative growthcompared to the triazine-resistant lines (Fig. 3). Such weakseedlings should be quickly out-competed by the triazine-resistant plants in a mixed population in the field.

The implications of the seed treatment for eliminatingatrazine-sensitive brassica plants presented in this studyclearly extend beyond the use of maternally inheritedtriazine resistance. Resistance to chlorsulfuron in soybeanscan be detected once seeds have been left to imbibe herbicidesolutions (Sebastian and Chaleff, 1987). We have alsodemonstrated that this approach can be used effectively toselect the herbicide-resistant progeny from Brassica napusplants that are either transgenic (Christey and Sinclair,1992) or have induced mutations (Conner et al., 1994) forchlorsulfuron resistance. For both of these latter sources ofchlorsulfuron resistance, overnight seed imbibition of 50 mgl−" chlorsulfuron resulted in healthy, resistant seedlings andstunted, chlorotic, sensitive seedlings that did not developbeyond the cotyledon stage (unpubl. res.).

Various innovative methods for hybrid seed productionusing herbicide resistance as genetic markers have beendesigned (Conner and Field, 1995). To date, the applicationof these novel approaches has required the use of herbicidesin either the seed production fields and}or the subsequentfields of commercial crops. The approach described in thispaper offers a more environmentally sound method foreliminating the contaminating herbicide-sensitive, non-hybrid seed. Imbibition of seeds in herbicides offers aconvenient approach for uniform exposure of plants tochemicals, and thereby reduces the problem of escapesresulting from environmental effects and errors associatedwith field applications. Such a simple seed treatment will bemore cost-efficient than field applications of herbicides andwill also contribute to the more efficient production ofhybrid seed.

ACKNOWLEDGEMENTS

We thank Jill Reader for her careful maintenance of theplant material during the course of this study, Robert

Lamberts for the photography and Adrian Russell forvaluable comments on earlier drafts of the manuscript.

LITERATURE CITED

Barsby TL, Chuong PV, Yarrow SA, Wu SC, Coumans M, Kemble RJ,

Powell AD, Beversdorf WD, Pauls KP. 1987. The combination ofPolima CMS and cytoplasmic triazine resistance in Brassica napus.Theoretical and Applied Genetics 73 : 809–814.

Beversdorf WD, Erickson LR, Grant I. 1985. Hybridisation processutilising a combination of cytoplasmic male sterility and herbicidetolerance. US Patent No. 4517763.

Christey MC, Earle ED. 1994. Field testing of atrazine-resistantcytoplasmic male-sterile-nigra broccoli (Brassica oleracea L. var.italica) obtained by protoplast fusion. Journal of Genetics andBreeding 48 : 33–40.

Christey MC, Makaroff CA, Earle ED. 1991. Atrazine-resistantcytoplasmic male-sterile-nigra broccoli obtained by protoplastfusion between cytoplasmic male-sterile Brassica oleracea andatrazine-resistant Brassica campestris. Theoretical and AppliedGenetics 83 : 201–208.

Christey MC, Sinclair BK. 1992. Regeneration of transgenic kale(Brassica oleracea var. acephala), rape (B. napus) and turnip (B.campestris var. rapifera) plants via Agrobacterium rhizogenesmediated transformation. Plant Science 87 : 161–169.

Chuong PV, Beversdorf WD, Powell AD, Pauls KP. 1988. Somatictransfer of cytoplasmic traits in Brassica napus L. by haploidprotoplast fusion. Molecular and General Genetics 211 : 197–201.

Conner AJ, Abernethy DJ, Dastgheib F, Field RJ. 1994. Brassica napusmutants with increased chlorsulfuron resistance. Proceedings ofthe New Zealand Plant Protection Conference 47 : 173–177.

Conner AJ, Field RJ. 1995. Herbicide-resistant crops: A new approachto an old problem or a radical new tool? In: McLean GD, EvansG, eds. Herbicide-resistant crops and pastures in Australian farmingsystems. Canberra: Bureau of Resource Sciences, 53–71.

Conner AJ, Meredith CP. 1989. Genetic manipulation of plant cells. In:Marcus A, ed. The biochemistry of plants, Vol. 15. Molecularbiology. Orlando: Academic Press, 653–688.

Kao HM, Brown GG, Scoles G, Se! guin-Swartz G. 1991. Oguracytoplasmic male sterility and triazine tolerant Brassica napus cv.Westar produced by protoplast fusion. Plant Science 75 : 63–72.

Sebastian SA, Chaleff RS. 1987. Soybean mutants with increasedtolerance for sulfonylurea herbicides. Crop Science 27 : 948–952.

Sokal RR, Rohlf FJ. 1969. Biometry: The principles and practice ofstatistics in biological research. San Francisco: Freeman.

Yarrow SA, Wu SC, Barsby TL, Kemble RJ, Shepard JF. 1986. Theintroduction of CMS mitochondria to triazine tolerant Brassicanapus L., var. ‘Regent ’, by micromanipulation of individualheterokaryons. Plant Cell Reports 5 : 415–418.