We used two genes, TMV-CP and PPI1 (pepper-PMMV interaction 1 transcription factor), to transform commercially important chili pepper (Capsicum annuum) inbred lines (P915, P409) by means of Agrobacterium co-culture. Eighteen independently transformed T0 plants were obtained. The most critical point in the pepper transformation protocol was the selection of shoots growing on calli—referred to as callus-mediated shoot formation (indirect shooting)—because shoots not grown from the callus (direct shooting from the wounded surface) developed into non-transformants. Selection of the correct right callus type also proved to be an important requirement for obtaining transformed peppers. Six different types of callus developed during the selection process. Shoots regenerated from two of these types, while one type regenerated significantly more shoots than the other types, suggesting that the capacity for shoot formation is callus type-specific. Although the transformation rate was low, transformation via callus-mediated shoot formation proved to be reproducible and was confirmed by Southern and Northern blot analyses. Based on the experimental data, we have succeeded in developing a new protocol for the selection and transformation of pepper and expect that it will be used in the future for pepper transformation.

A new selection method for pepper transformation: callus-mediated shoot formation

Genetic Modification of Plants

Direct shoot organogenesis on shoot apex from seedling explants of Capsicum annuum L.

A MADS box gene AGL20/SOC1 is a main integrator in Arabidopsis flowering pathway whose structure and function are highly conserved in many plant species. A soybean MADS box gene GmGAL1 (Glycine max AGAMOUS Like 1) as a homolog of AGL20/SOC1, was cloned from soybean cultivar Kennong18 and its function was investigated in transgenic Arabidopsis lines. Sequence comparisons showed that the closest homolog gene to GmGAL1 is AGL20/SOC1 in Arabidopsis and VuSOC1 in Vigna unguiculata. We investigated the expression level of GmGAL1 using quantitative real-time PCR, and the result showed that GmGAL1 was regulated by a circadian mechanism and its expression oscillated at a cycle of 24 h. The expression level of GmGAL1 was fluctuated in diverse tissues/organs and developmental stages. Considering its expression can be detected in different tissues throughout the life cycle and its protein localized in cytoplasm in Arabidopsis protoplasm, we proposed that GmGAL1 may be a multifunctional gene in the context of the soybean development. Ectopic expression of GmGAL1 in Arabidopsis enhanced flowering under long-day condition and partially rescued soc1 late flowering type.

Cloning and expression analysis of GmGAL1, SOC1 homolog gene in soybean.

A MADS box gene AGL20/SOC1 is a mainintegrator in Arabidopsis ﬂowering pathway whose struc-ture and function are highly conserved in many plantspecies. A soybean MADS box gene GmGAL1 (Glycinemax AGAMOUS Like 1) as a homolog of AGL20/SOC1,was cloned from soybean cultivar Kennong18 and itsfunction was investigated in transgenic Arabidopsis lines.Sequence comparisons showed that the closest homologgene to GmGAL1 is AGL20/SOC1 in Arabidopsis andVuSOC1 in Vigna unguiculata. We investigated theexpression level of GmGAL1 using quantitative real-timePCR, and the result showed that GmGAL1 was regulated bya circadian mechanism and its expression oscillated at acycle of 24 h. The expression level of GmGAL1 wasﬂuctuated in diverse tissues/organs and developmentalstages. Considering its expression can be detected in dif-ferent tissues throughout the life cycle and its proteinlocalized in cytoplasm in Arabidopsis protoplasm, weproposed that GmGAL1 may be a multifunctional gene inthe context of the soybean development. Ectopic expres-sion of GmGAL1 in Arabidopsis enhanced ﬂowering underlong-day condition and partially rescued soc1 late ﬂower-ing type.Keywords Soybean (Glycine max [L.] Merr.) GmGAL1 FloweringIntroductionMADS-box genes belong to a large multifunctional familywhich controls the identity of the apex meristem, devel-opment of lateral organs, and ﬂowering time of plants [1–8]. In Arabidopsis, AGAMOUS-like (AGL) genes belong toa sub-family of MADS-box genes which play importantroles especially in regulating ﬂowering pathways. Forexample, FLOWERING LOCUS C (FLC, AGL25) acts as arepressor of ﬂowering [9–12]. APETALA1 (AP1, AGL7)promotes ﬂowering and controls the identity of meristemand sepals [13, 14] and AGL12 regulates both ﬂoweringtime and root development [15].Another AGL gene, SUPPRESSOR OF OVEREX-PRESSION OF CONSTANS (SOC1), is a critical integratorof different ﬂowering pathways which promotes ﬂoweringin Arabidopsis [16–20]. Its expression was detected inseveral organs include leaves, root, steam apex, etc. [19,21]. In development stages, mRNA abundance of SOC1increases after seed germination and further accelerates inﬂower meristem during ﬂower organs transition [19, 21].Ectopic expression of the SOC1 gene is sufﬁcient to inducethe early ﬂowering in both long-day and short-day condi-tions [22], while lose-of-function of SOC1 results in lateﬂowering. Although soc1 mutant show obviously lateﬂowering time phenotype, it’s ﬂower organs developalmost normally because it is functionally redundant withAGL24 and SHORT VEGETATIVE PHASE (SVP) in thecontrol of ﬂower organ identity [21, 23].SOC1 genes are widely expressed in various tissues.Another SOC1 like gene ETL (Eucalyptus TM3 Like,

Cloning and expression analysis of GmGAL1, SOC1 homolog gene in soybean

Two transgenic pepper plants were obtained from 255 seed explants that were infected with Agrobacterium LBA4404 (pGA1209). One of them (PT2) showed morphological change, such as dwarfism and early flowering by the constitutive expression of the rice OsMADS1 gene. The in vitro condition of the plant regeneration has been optimized from hypocotyl explants on a MS medium that was supplemented with zeatin 3 mg/L, IAA 0.3 mg/L for shoot induction. The optimal rooting condition was at NAA 0.3 mg/L. The transformation frequency was 0.8% from the total hypocotyls. DNA and RNA hybridization analyses showed that the introduced gene was integrated and stably expressed in regenerated plants.

Lee Kw

Papers

Constitutive expression of rice MADS box gene using seed explants in hot pepper (Capsicum annuum L.).