Effect of genotype and medium composition on flax Linum usitatissimum L. anther culture

Identification of responsive genotypes and development of efficient protocols are the prerequisite to an effective doubled haploid production system in applied breeding programs. Evaluation of 16 low linolenic flax (Linola) genotypes/populations with diverse genetic backgrounds from a Linola breeding program using A₂₂C medium containing 9% sucrose (A₂₂C-9) led to the identification of a number of responsive genotypes. For 96-3-F₁ hybrid, callus induction was greater in modified NLN medium containing 12% sucrose (NLN-12) than in A₂₂C-9. But there was no difference in shoot regeneration between NLN-12 and A₂₂C-9. For 96-45-F₁ hybrid, there was no difference in callus production between the two media. However, A₂₂C-9 had a greater shoot regeneration than NLN-12. In comparison to sucrose, lactose was found to increase callus induction from anthers for all three genotypes tested. However, the effect of lactose on shoot regeneration appeared to be genotype-dependent.     

Pyramiding of alleles with different rust resistance specificities in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.

Transgenic flax plants expressing flax rust resistance specificities conferred by L ² and L ¹⁰ alleles of L locus were produced through Agrobacterium tumefaciens-mediated transformation of hypocotyl segments or anther culture derived-calli. Transgenic plants were characterized by PCR amplification and Southern hybridization. Homozygous transgenic lines containing a single locus of effective transgene were isolated by consecutive progeny analyses of transgenic plants. In addition, homozygous lines were directly obtained from transformation of haploid cells followed by spontaneous or artificial chromosome doubling when anther culture derived-calli were used as the explants. All transgenic plants containing L ² transgene expressed L ² flax rust resistance specificity and had unambiguous infection type (IT) “0” (immune) reactions to flax rust race 22, which is virulent to endogenous L ⁶ and K ¹ genes present in the untransformed Linola™1084. Transgenic plants containing L ¹⁰ transgene exhibited various levels of resistance to flax rust race 191. Five plants had IT “fleck” (immune) reactions, similar to the reactions with the L ¹⁰ rust differential line, Bolley Golden Selection. The enhanced resistance to rust race 191 in these transgenic plants was attributed to the expression of L ¹⁰ rust resistance specificity. Further evaluation of the transgenic plants containing L ² or L ¹⁰ transgene to flax rust race 258 and race 247 respectively showed that the endogenous rust resistance specificities were not modified. The implication of this study in producing transgenic flax plants with multiple resistance specificities and for crop improvement using molecular breeding strategy is discussed.     

Effect of medium osmotic potential on callus induction and shoot regeneration in flax anther culture

Development of an efficient and cost-effective doubled haploid production system in flax (Linum usitatissimum L.) is the prerequisite for the application of doubled haploid technology in a practical breeding program. Pre-culture of anthers on a medium containing 15% sucrose for 2–7 days before transfer to the same medium containing 6% sucrose for a total of 28 days culture period significantly increased shoot regeneration for all four genotypes evaluated. Moreover, pre-culture of anthers on medium containing 15% sucrose for 2–7 days was sufficient to dramatically reduce the frequency of shoot regeneration from somatic tissues and thereby to increase the frequency of microspore-derived plants in flax anther culture. Furthermore, replacing 15% sucrose with 6% sucrose and 9% polyethylene glycol (PEG), or 3% sucrose and 12% PEG, in pre-culture medium did not significantly affect callus induction and shoot regeneration. The results indicate that sucrose may act as carbon/energy source as well as an osmotic regulator in flax anther culture. Sucrose as an osmotic regulator may be replaced by a non-metabolizable osmoticum: PEG. The implication of this study in flax anther culture and breeding is discussed.     

Response of flax genotypes to doubled haploid production

Forty-four flax genotypes with a diverse genetic background were evaluated for anther culture response using a standard anther culture protocol in order to determine the feasibility to initiate a routine haploid production system in applied breeding programs. A strong genotype effect on callus induction and shoot regeneration in anther culture was found in this study. A number of genotypes, including two low cadmium content lines 96-11785 and 96-11826, a high oil content line 96-22109 and a high linolenic acid content line M 4919 were identified as highly responsive. The impact of the findings in this study on flax breeding was discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of sucrose concentration on elongation of shoots from flax anther culture

Anther culture is considered as the most successful method of producing doubled haploid plants in flax. The efficiency of shoot regeneration from anther culture has been improved dramatically by optimizing culture temperature and callus induction medium. However, shoot elongation has become increasingly the limiting factor for further improvement of the overall efficiency of doubled haploid production. The effect of sucrose con- 21 centration on shoot elongation was investigated in this study. The medium containing 10 g l sucrose produced longer and more vigorous shoots than the same medium containing other concentrations of sucrose. The possible physiological basis of sucrose on shoot elongation in flax was discussed.     

Identification of microspore-derived plants in anther culture of flax (Linum usitatissimum L.) using molecular markers

The microspore origin of anther-culture-derived plants of flax was determined using inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Polymorphic fragments between the two parents of the F₁ donor plants were identified and their segregation patterns in anther-culture-derived plants were used to elucidate the origin of those plants and to determine the degree of independence of plants regenerated from the same callus. Using one ISSR primer (UBC 889) and two RAPD primers (UBC 556 and 561), 12 out of 16 plants were unequivocally identified as being derived from microspores. Plants derived from the same callus had identical PCR patterns at five polymorphic loci and thus were likely derived from the same microspore. Therefore, it is proposed that the number of calli forming shoots be used to describe the anther culture efficiency in flax. Received: 3 February 1998 / Revision received: 8 June 1998 / Accepted: 8 July 1998