The days anthesis in relation to the floral shapes , pollen size , and S-RNase gene identifying in self-incompatibility of wild species

Flowering time of tomato (Solanum.chilense) which is a self-incompatibility wild species flowers was determined using a series of landmark days. We established a correlation between the floral shapes and the days before anthesis. The model of tomato flower development schedule was then used to analyze the size of bud, anther, style, and the pollen scanning electron microscope (SEM) in the days before anthesis. Two new S-RNase genes of the Solanum .chilense which was the stylar SI genes were cloned. The structure of the two S-RNase was also analyzed. Finally, the different topology of the phylogenetic trees produced using available S-RNase sequences suggests that the S-RNase have different evolutionary histories.


INTRODUCTION
Research in plant developmental biology has progressed very rapidly in the past decade due to the application of molecular biology techniques, the isolation and characterization of various developmental mutants, and the cloning of genes specifically involved in different stages of plant development.It has been established that floral organ development is controlled by the differential expression of tissue-and organ-specific genes (Meyerowitz, 1998;Theissen et al., 2000).Such genes may be involved in organ/tissue differentiation by triggering the regulatory cascade of key genes controlling the identity of floral organs, that is, the "ABC" model of floral development (Coen and Meyerowitz, 1991;Weigel and Meyerowitz, 1994;Ng and Yanofsky, 2000).The ontogeny of tomato (Lycopersicon esculentum cv.sweet cherry) flowers was subdivided into 20 stages using a series of landmark events (Vladimir Brukhin et al., 2003).However, these models are not suitable to the research of pollination biology.Self-incompatibility (SI) is a genetic *Corresponding author: E-mail: shuainanhai81517@sohu.com.mechanism found in many flowering plants that acts to inbreeding (De Nettancourt, 2001).In the best-reduce the negative fitness effects associated with understood examples, SI is controlled by a single genetic S locus with many S alleles: styles reject pollen grains when both express the same S specificity.So, the problem that whether the self pollen is rejected by itself style is the key problem .The better method to answer this problem is the pollination biology.In this paper, we established a correlation between the floral shapes and the days before anthesis.And the model of tomato flower development schedule was then used to analyse the size of bud, anther, style, and the pollen EMS in the days before anthesis, the most important is that we know which day of the days before anthesis is the day of the viable pollen.
Using our model of tomato flower development schedule on tomato flower development, we have been able to identify critical stages at which major pollens is the viable pollen.We also show that the flower development schedule can be used to analyse self-incompatibility of wild species pattern of pollination in tomato flowers.And we cloned two new the S-RNase genes illustrated self-incompatibility (SI).

Materials
Seeds of self-incompatibility (SI) S. chilense (LA0130) and selfcompatibility (SC)S.habrochaites (LA0094) were obtained from the Charles Rick Tomato Genetics Resource Center at the University of California Davis http://tgrc.ucdavis.edu/index.aspx and were germinated in peat pellets and seedlings with three to four leaves were transferred into 15 cm plastic pots containing the perlite :peat (1:1).The plants were grown in greenhouse under a thermoperiod of 26/20°C (day/night).Plants were supplied every week with a commercial fertilizer.

Flower development schedule define
Inflorescence and flower sampling were performed on 2-month-old plants after the production of three inflorescences.Then flower development schedule was defined.

Scanning electron microscopy
Samples were fixed in 69% acetone/29% ddH2O/2% glutaraldehyde (100%) for 24 h at 4°C , washed, dehydrated with 30, 50 and 70% ethanol for 15 to 20 min separately and samples were then dried as following tert butyl alcohol(TBA) , ddH2O, and ethanol concentration grads (Table1).Every grad needs 15 to 20 min and sputtered with gold.Specimens were examined by SEM (NOVA NanoSEM 230) at an accelerating voltage of 5.00 kV, magnification of 1000 ⅹ, electron microscope detector of LVD.Images were saved as digital files.

Cloning and DNA sequencing
PCR products were separated in 1% agarose gels, and cloned into pMD18-T vector system (TaKaRa) for sequencing.S-RNase genes identified in the present study are registered in GenBank database as ZS4 (JN226563), ZS5 (JN226564).All evolutionary analyses and alignments were performed using DNAMAN and ClustalW.

RESULTS AND DISCUSSION
Defining flowering time S. chilense belongs to the Solanaceae family.It is a plant featuring a sympodial growth habit of the main shoot and axillary branches; after producing eight to ten leaves, the vegetative meristem transformed into the inflorescence meristem.The next vegetative apex was formed by the lateral bud in the axil of the leaf closest to the apical meristem.Later, the growth of this meristem pushed the inflorescence meristem to the side.The inflorescence meristem ultimately appeared as a lateral monochasial inflorescence, which is a dichotomously branched scorpioid uniparous cyme of six to ten flowers on each branch (Figure 1).Flowers contained five sepals, alternating with five petals, five anti-sepalous stamens forming a cone around the style (lateral cohesion of anthers by the interweaving of hairs) and two fused carpels forming a style.The flower was partly gamosepalous.In mature tomato flowers, stamens were shorter than the style, and the anthers formed a tube around the style.
S. chilense (LA0130), the days before anthesis and the days after anthesis is presented in Figure 2. The developmental process has been subdivided into several days (the days before anthesis and the days after anthesis).The following sections detail the comparative development of flower in relation to the days before anthesis and the days after anthesis (Figure 2).

The size of floral organ at the days before anthesis
The very early stages of flower development (ranging from 200 to 1,000 mm in length) have already been described (Sawhney and Greyson, 1972;Chandra and Sawhney, 1984;Rasmussen and Green, 1993) and the size of floral organs summarized as 20 stages (Vladimir et al., 2003).The data reported here that showed in relation to the days before anthesis and the bud forms, the bud size, the anther size, the style length (Table 2).
Comparative the days before anthesis in relation to the bud size, the anther size, the style length showing that the bud size and the anther size continued to grow not to stop from the 7 days before anthesis to the 0 day before anthesis.

Pollen SEM
The size of pollen increased from the 7 days before anthesis to the 3 days before anthesis, and then decreased until the 0 days before anthesis (Figures 3 and  4).The dehydration of pollen will happen at the 3 days before anthesis.Tomato pollen development is presented in Figure 5.The developmental process was subdivided into these stages, as follows: stage 1 pollen mother cell, stage 2 tetrad, stage 3 free microspores, stage 4 uninucleate microspores, stage 5 immature pollen grain, stage 6 mature pollen grain.The dehydration process of pollen happened from the stage 5 to the stage 6.So the 3 days before anthesis, the dehydration process of pollen happened from the stage 5 to the stage 6.The pollen have became the mature pollen before the 2 days before anthesis in Solanum chilense (LA0130).

The pollen vitality of the days after anthesis
Testing techniques of plant pollen viability were summarized as follows: staining method, germination method and pollination in field method, etc.But the staining method was the convenient and efficient method.In this paper, the pollen vitality was tested by the acetocarmine method.The pollen exists the vitality from the 0 days after anthesis to the 6 days after anthesis.Pollination can The 4 days after anthesis, The 3 days after anthesis, The 2 days after anthesis, The 1 day after anthesis, The 0 day after anthesis.
be done in these days.

Pollination with self pollen
Pollen tube growth in self pollinations was quantified, as shown in Figures 7 and 8.After 48 h, pollen tubes reached the ovary in self pollinations of a SC accession LA0094 and didn't reach the ovary in self pollination of a SI accession LA0130 as predicted.In the SI accession LA0130, the pollen tube almost stopped growing from 6 to 24 h, started growing after 24 h, but don't reached the ovary.In the SC accession LA0094, the pollen tube constantly grew, reached the ovary and fruiting finally.
In the past study, the ontogeny of tomato (Lycopersicon esculentum cv.sweet cherry) flowers was subdivided into  20 stages using a series of landmark events (Vladimir et al., 2003).The 20 stages were used to study the ontogeny of tomato, which was not suitable for use in the studying of pollination biology.In this study, we detailed the comparative development of flower in relation to the days before anthesis and the days after anthesis and established the model of tomato flower development schedule, the correlation between the floral shapes and the days before anthesis, the days after anthesis.And then the model of tomato flower development schedule was used to analyse the size of bud, anther, style, and the pollen SEM in the days before anthesis.Also, we established the correlation between the pollen vitality and the days after anthesis and used to study the pollination biology of the self-incompatibility of wild species.And we knew which day was the best day for the pollination.In addition, this paper describes the identifying of the S-RNase genes of the Solanum.chilense(LA0130) which will be useful to us in future study.
In conclusion, we have constructed a detailed reference model of the correlation between the floral shapes and the days before anthesis and the pollen vitality of the days after anthesis for the later study of the pollination and especially for self-incompatibility of wild species pollination.

Figure 3 .
Figure 3. EMS of Pollen in relation to the pollen form of the days before anthesis (dba).

Figure 4 .Figure 5 .
Figure 4.The size of Pollen in relation to the days before anthesis (dya).

Figure 6 .Figure 7 .
Figure 6.The pollen vitality of the days after anthesis

Figure 8 .
Figure 8. SI and SC pollen tube length in self pollinations.

Table 2 .
The size of floral organ at the days before anthesis.