Bolting and Flowering Response of Lactuca georgica, a Wild Lettuce Relative, to Low Temperatures

To learn about the phenological adaptation of Lactuca georgica Grossh., a wild 
relative of domesticated lettuce, we studied seed sampled accessions obtained 
from individual plants at 19 locations throughout six regions in Armenia, and 
from two natural populations in Dagestan (Russian Federation) collected as bulk 
samples. The effects of various vernalization treatments on time to bolting, 
flowering and seed production time were investigated during four successive 
years at different growth stages of L. georgica plants. We demonstrate that low temperatures 
play a major role in stimulating the reproduction process of L. georgica plants. Our results 
would suggest that for L. georgica: 
1) There is an obligatory (or nearly so) vernalization requirement; 2) Plant 
age, vernalization duration, and genotype of original sample have a role in 
bolting and flowering regulation; 3) Some plants behaved as typical annuals, 
responding to vernalization treatment at the seedling stage, but, most did not; 
4) Four months of vernalization could be adequate to reach bolting in plants 
with a developed vegetative rosette, for most—but not all—samples; 5) In order 
to find the best solution for stimulating the reproductive process of multiple 
genotypes, it seems that further study should focus on about 4 - 6 months of 
vernalization at 4°C applied to plants of about 10 - 22 months old vegetative 
rosettes, with controlled post-vernalization condition; 6) L. georgica germplasm could be 
used as a source for delayed bolting in breeding of domesticated lettuce 
varieties.


Introduction
The genus Lactuca L. [Compositae (Asteraceae), tribe Cichorieae, subclade Lactucinae [1] is comprised of 100 [1] to 148 species [2], which are mainly distributed in the Northern Hemisphere ( [3], and literature cited therein). The domesticated species in the genus, Lactuca sativa L. (lettuce), is one of the most important and widely distributed leafy vegetables around the world. Domestication has resulted in limited genetic variation in the crop making it vulnerable to diseases, pests, and environmental stresses.
In recent years, we have performed extensive studies on the characterization of wild Lactuca spp. originating from Southwest Asia, the center of diversity for wild species closely related to domesticated lettuce (Wild Lactuca Relatives, WLRs) [4]. Unique new collections of L. serriola, L. aculeata Boiss., L. georgica Grossh., and L. altaica Fisch. & C. A. Mey. (four of the seven wild species, according to previous literature [4] [5], in the primary lettuce gene pool, LGP-1), and L. saligna (in LPG-2) from Israel and Armenia were studied, as well as a few samples previously collected from Jordan, Turkey and other Mediterranean and European countries. The objectives of our research are related to the identification, collection, characterization, conservation, and sustainable use of these rich genetic sources for lettuce improvement. These studies included eco-geographical distribution [6] [7] [8], genetic, morphological and phenological diversity [9] [10] [11] [12] [13], downy mildew resistance [10] [14] [15] [16] [17] and variation of biologically active sesquiterpene lactone contents [18] [19] [20] [21]. Obtained results strongly support the use of these species specifically and WLRs in general as rich genetic sources for lettuce improvement.
Prior to 2009, in world gene bank collections of wild Lactuca spp., L. georgica was represented by only a single sample [24] [25], "LAC 327". Consequently, L. georgica was not studied by lettuce breeders and crop evolutionists [3]. Thus, we strove to increase the number of available L. georgica samples for comparative genetic and physiological studies.
Plants originating from a seed sample of the L. georgica "LAC 327" were grown in 2009 and morphologically characterized alongside of wild Lactuca spp. samples representing (according to Lebeda et al. [5]) the LGP-1 (L. aculeata, L. serriola, L. dregeana, and L. altaica), LGP-2 (L. saligna), and the section Mulgedium (Lactuca tatarica (L.) C. A. Mey.). Field regeneration was conducted at the Institute of Evolution (IOE), Haifa University (HU), Israel and followed all standard seed multiplication protocols, except that seeds were not vernalized. All plants of the L. georgica sample did not bolt, even after all plants from the other Bolting resistance is an important breeding aim of vegetative crops [26].
Therefore, information on the phenology of L. georgica, and, in particular, on the environmentally mediated response, is of great importance. It is also important to learn if all L. georgica germplasm is indeed biennial, which is a commonly-accepted "fact" that has been cited for over one hundred years in the literature ( [23], p. 80), but without any experimental data to supported it. A biennial plant, by definition, completes its life-cycle during two years: in the first year, plants generally make only leaves and other vegetative structures, while in the second year they flower, produce seed and die. The change from vegetative to reproductive growth is a key developmental switch in flowering plants [26]. Environmental conditions such as temperature affect survival, growth, and fitness, particularly during key stages such as seedling growth and reproduction ( [27], and literature cited therein). The timing of flowering in both wild and crop plants is a fundamental aspect of adaptation. The control of time to flowering has been studied in numerous species and is quite complex. Under normal circumstances, external cues such as low temperature (vernalization) and light (duration of exposure and intensity) are the prime factors that determine when plants will blossom [28]. The requirement for vernalization has been studied in many wild (and derived domesticated) species with both monocarpic habit (those that flower, set seed and die) and polycarpic species (perennials that can flower repeatedly over many years) showing a requirement for vernalization [29].
In several species, the response to vernalization varies with plant age. The requirement for vernalization usually increases or decreases linearly with respect to time. An increased requirement is found in species with a juvenile phase preceding the inductive phase. A decreased requirement is found in species with a quantitative response to vernalization, since, in these species, floral induction proceeds gradually with age even in the absence of a cold period [30]. In the present study, we examined the effect of low temperatures on the bolting and flowering time of germinating seeds and on different ages of the vegetative rosette of L. georgica plants. Experiments were performed during four successive growing seasons. From this, we hoped to gain innovative insights in the fields of phenological adaptation and germplasm exploitation of this WLR, aiming to improve our plant genetic resources (PGR) use efficiency for both basic research and potentially novel sources for breeding programs with domesticated lettuce.

Plant Material
A total of 121 wild L. georgica samples were used in this study ( Table 1). The majority of original seed samples were collected from individual plants at 19 unique locations throughout six regions in Armenia (Figure 1(A) and Figure  1 (Table 1).
Various observations supported species identity of the L. georgica samples as following: 1) morphological and developmental evaluation, in their native habitats, according to botanical keys and some basic descriptors for wild Lactuca spp.

Experiments and Growing Conditions
Different vernalization treatments were evaluated at the  (Table 1(c)). Vernalization treatments were applied to either germinating seed or older plants. The experiment in 2012 on older plants and 2012-2013-iv experiment used plants generated from the 2010 and 2011 experiments, respectively, on germinated seed. The plants used for these two experiments had previous exposure to vernalization as germinated seed. We assumed that these plants did not "remember" the old cold period, thus results can be attributed to the new treatments that included the various vernalization durations as described above. The plants used in the control treatment were the exception to this; they were gener- In other experiments, where vernalization treatments were applied to plants of different ages of the vegetative rosette, we used plants from the previous experiments or other control plants. These kinds of experiments were not optimal because the start and end vernalization date were staggered, however they did provide information if old plants with various ages can be stimulated to bolt.

2010 Experiment
In a preliminary experiment in 2010, five different combinations of duration and vernalization temperature were applied to just germinating seeds (48 h after imbibition), each included five plants (replicates) from four seed samples representing four populations from the 2009 collection (see Table 1(a)). Treatments were as follows: 1) 27 days at 4˚C; 2) 27 days at 1˚C; 3) 37 days at 4˚C; 4) 37 days at 1˚C; 5) control. To sum up, a total of 25 plants from each sample and a total 100 plants were included in the experiment. All vernalization treatments ended on May 17, 2010. Plants were then moved to a lath house. The temperature from May 17 to May 30 ranged from −0.9 ˚C to 24.7˚C, with an average temp. of 10.7˚C. The high temp. for June was 28.7˚C on June 27. Day-length during this period was above 15 h.

2011 Experiment
Three vernalization durations were performed at 4˚C using just germinating seeds (48 h) from the same four seed samples that were used in the 2010 experiment (see Table 1(a)): 1) 37 days with four replicates from each sample; 2) 44 days with four replicates; 3) control, i.e. without vernalization, with three replicates. A total of 11 plants from each sample, for a total of 44 plants were included in this experiment. All vernalization treatments ended on Aug. 7, 2011, then plants were moved for 14 days of post-vernalization adaptation in a growth room at 15˚C under 18 h-daylength. After 14 days, the temperature was raised to 18˚C under 18 h daylength. The plants were under these conditions for the remainder of the experiment.

2012 Experiment
A period of 120 days (4 months) of vernalization at 4˚C was applied to a total of 12 plants, three plants (replicates) each from the same four seed samples that were used in the 2010 and 2011 experiments (see Table 1(a)), which had re-   Table 1(a)), that were used in previous experi-  bolted. Note that these two samples were originally collected at higher elevation as compared to the two samples with bolting plants (see Table 1(a)).

2011 Experiment
Only one out of four plants (25%, Figure 2

2012 Experiment
Of the 21.6 months old plants, a total ten out of the 12 plants bolted after the four months vernalization at 4˚C. All three plants (100%, Figure 2 Aug. 05, with an average of 60 dpv. First seed production ranged from July 23 to Aug. 9, averaged 74 dpv.

Discussion
This study is the first investigation of phenological adaptation of L. georgica natural populations and individuals from Armenia and Dagestan, specifically vis-a-vis vernalization requirements. It was shown that low temperatures play a major role in stimulating the reproductive process of L. georgica plants. Our results would suggest that: 1) L. georgica has an obligate (or nearly so) vernalization requirement since bolting occurred only in a single plant which was not exposed to low temperatures. The single control plant from sample W6-37160-10 that did bolt was a well developed plant, approximately 20 months of vegetative age (2012-2013-iv; Figure 2(F)). 2) Plant age, vernalization duration, and genotype of original sample have a role in bolting and flowering regulation of L. georgica plants. However, further experiments are required to define the minimal requirements for floral initiation of L. georgica. 3) Some samples of L. georgica appear to behave as non-obligate typical biennials. Bernier et al. [33] indicated that biennial plants with an obligate vernalization requirement normally undergo a juvenile phase during which they are insensitive to low temperatures.
However, some of our tested germplasm, in which germinating L. georgica seeds did respond to the vernalization treatment, do not fit this scheme (Figure 2(A) and Figure 2 Figure  2(F)) experiments bolted after four months of vernalization. However, a longer vernalization duration is needed for some samples, even after attaining a well-developed vegetative stage. 5) To find the best solution for stimulating the reproduction process of L. georgica plants from populations that represent different climatic and edaphic environments, it seems that further study should focus on about 4 -6 months of vernalization at 4˚C applied to plants with about 10 -22 months of the vegetative rosette, with controlled post-vernalization conditions. Generally, high temperatures and short days may counteract the inductive action of cold temperatures on a plant's bolting and flowering [33]. 6) Due to their vernalization requirement, L. georgica germplasm may be used as a source for delayed bolting in breeding domesticated lettuce varieties. High temperature induces early bolting and flowering in lettuce [34]. So, increased temperatures from global climate change pose great challenges for lettuce production. Therefore, it is urgent to study the genetics and molecular mechanism of late bolting and flowering in lettuce by using WLRs to identify novel genes and alleles in known genes that were eliminated following lettuce domestication.
Along with the potential to use L. georgica as a source for delayed bolting, the biochemical features [19] [21], and downy mildew resistance [16] point to the uniqueness of this species. Even though recent results indicate that L. georgica probably belongs to the LGP-2 [9] [35], we suggest that it should be considered as an attractive germplasm resource for domestic lettuce breeding programs.
Clearly, its uniqueness justifies identification and collection of additional samples from multiple locations throughout its geographic distribution.
Due to the limited extent of prior research on L. georgica, this study is by necessity exploratory, with the various experiments designed sequentially, and somewhat ad hoc, limited by the availability of seed samples from populations across the species' range, and by the huge amount of work to perform the type of experiments in the present study. It is hard to draw definitive statistically-supported conclusions about the contributions of plant age, vernalization time, and genotype to bolting probability and time to flowering from these results. Nevertheless, a pretty clear basic picture emerges, indicating that for most genotypes vernalization induces bolting with any consistency only in older plants, and there is evidence of genotypic variability in this behaviour. Clearly, more systematic follow-up research on L. georgica using germplasm from a wider geographic range is warranted.