DEVELOPMENTAL CONTROL OF SOME PHYSIOLOGICAL FACTORS ON REPRODUCTIVE BIOLOGY AND RUDIMENTARY EMBRYOS PHENOMENON IN CARROT SEEDS

This study was aimed to investigate delayed germination and immature embryos of carrot seeds; This research was implemented at vegetable field of College of Agricultural Engineering Sciences - University of Baghdad during two sequent seasons (fall and spring). The experiment was conducted using factorial within Randomized Complete Block Design arrangement with three factors and replicates (3X3X2). The aqueous barley sprouts extract (B 0 , B 1 ) (0, 100 g.L -1 ) represented the first factor. Trehalose (T 0 , T 1 , T 2 ) (0, 50, 75 mmole.L -1 ) represented the second factor. Calcium (C 0 , C 1 , C 2 ) (0, 1, 2 ml.L -1 ) represented the third factor. Results showed the effectiveness of third order interaction treatment B 1 T 2 C 1 in increasing germination speed and percent for the seeds of royal umbel (92, 97%); secondary umbel (90.67, 94.67%); and tertiary umbel (75.68, 86.33%). The mentioned treatment increased embryo length for the seeds of primary (royal) umbel (1628.3 µm), secondary umbel (1620.3 µm); and tertiary umbel (874.7 µm ). in compare with the lowest percents that found in B 0 T 0 C 2 treatment.


INTRODUCTION
Carrot Daucus carota that belongs to Apiaceae; has very sophisticated reproductive biology because of its distinctive features that could be summarized by First; umbels consequent appearance, Second; carrots protandrous flowers (20), and finally; carrot rudiment embryos (23).The main cause of this problem is a defect in carrot embryogenesis that illustrated by the growth and enlargement of endosperm over embryo.As a results; carrot seed embryo forms 2-3% only from endosperm (21).What mentioned prior leads to cause a cascade of different problems; their final result is producing seeds with different qualities and large percent of weak seeds (23).Sprouting grains sharply increase GA 3 and rapid decrease in ABA levels, which could be a good reason to activate embryogenesis in carrot seeds (24).Moreover; they considerably increase nutrients bioavailability, i.e. breaking down proteins to amino acids, starch to sugars, and fats to fatty acids (17).Hence, their aqueous extracts could be sprayed on plant and have a good impact (3,4,5).Trehalose has many functions in plant such as, carbon metaboliztion, plant protection from abiotic stresses (2 , 6, 13, 22) and signalization (8,11,16).In addition, it has proven that trehalose organizes florigen transporting to shoot tips in order to evocate floral induction.Moreover; trehalose activates SPL gene that also stimulates flowering (Cho) (22).Eastmond et al (9) demonstrated that trehalose metabolism is necessary for embryogenesis stages completion.Calcium an essential element in plant reproductive biology since it directly affects fertilization.It considers a directional cue for pollen tube.Even more; calcium establishes the polarity of pollen tube.As for the ovary and ovules; calcium abundance in micropyler region and embryo sac attracts the pollen tube entry (7,10,12).Consequently, this study was aimed to target carrot seeds embryogenesis by using different treatments (sprouts aqua extract, trehalose, and calcium) that are targeting the same issue but with different mechanisms of action.

MATERIALS AND METHODS
This experiment was conducted during two sequent seasons, fall -first growth cycle (roots production), and spring -second growth cycle (seeds production) at researches station (A) College of Agricultural Engineering Sciences, University of Baghdad (Al-Jadiryah).Table 1 shows the chemical and physical characteristics of the soil for the two seasons.For the first season; the seeds of carrot var.Nantes were sowed on rows on terraces in 15/September.The field was under drip irrigation system.The seeds were planted in a plant density 1,000,000 plants.ha - .The entire plots harvested after 115 days of the planting date.The produced stecklings (roots) stored in a dark place at 4ºC for 28 days to induce vernalization (21).After that (the second season); carrot stecklings transplanted on terraces with two rows.The spacing between one plant and another was 0.3 m.Mineral fertilizer was added as recommended for carrot plants (120 kg.ha -1 , 120 K 2 O 5 kg.ha -1 , 40 K 2 O kg. ha -1 ) to all plots before transplanting (1).The experiment was implemented using randomized complete block design with factorial arrangement (2X3X3) with three replicates.Spraying barley aqueous sprouted grains extract was represented the first factor with two levels (0, 100g.L -1 DW) which symbolized (B 0 , B 1 ).The second factor is spraying with three levels of trehalose (0, 50, 75 mmol.L -1 ) which symbolized (T 0 , T 1 , T 2 ).The third factor is spraying with three levels of calcium (0, 1, 2 ml.L -1 ) (as chelated calcium 30% Ca), which symbolized (C 0 , C 1 , C 2 ).The plants foliarly sprayed for three times.For the first season (roots production); The first spraying was ten days after thinning and the duration between one spraying and another was 15 days.For the second season (seeds production); the first spraying was after emergence completion.The period between sprayings was 30 days.

Table 1. Physical and chemical characteristics of the soil for the two seasons
Barley aqueous sprouted grains extract was prepared by hydroponically germinating 100 g of barley grains, var.Ebaa class 265, (until radical penetration stage).After that, the germinated grains blended in an electric blender until the ingredients were mixed well.
Then the solution was filtered by whatman filter paper 42 pore size and the volume was completed to 1 liter.The extract was sprayed on the plants directly after preparation.An aqueous extract was prepared from the quiescent barley grains (for the purpose of comparison and calculating the nutritional bioavailability).Table 2 shows the chemical and physical properties and conversion ratio of barley aqueous sprouted and grains extract.
The Study traits were Determination of the following; 1-Anatomical (histological) traits: Anatomical traits were carried out on carrot seeds in Nematode Laboratory/Plant Protection Department/College of Agricultural Engineering Sciences.Carrot seeds were placed in water for two hours, and then longitudinal sections of the seeds were taken from the first third of the seeds (figure 1) under an optika electron microscope.Then dye it with a salt solution of 2,3,5-Triphenel Tetrazolium Chloride, commercially known as tetrazolium salt or TZ, at a concentration of 1 g. 100 ml -1 distilled water and leave it in the dark for two hours at room temperature for the reaction to complete (15), as the salt dyes.The living tissue in the seed (embryo) is colored red, making it easy to distinguish from the endosperm, which is very similar in color.It was then examined under an electron microscope using a graduated lens.The results of Table (3 B) indicated that there were significant differences in second order interaction between the aqueous extract of barley sprouts and trihalose.(B 1 T 2 ) produced the longest embryo for the seeds of primary, secondary, tertiary umbels (1367.8,1315.8 and 773.8 µm) respectively.With reference to the interaction between aqueous germinated barley grains extract and calcium (Table 3B); B 1 C 1 showed the longest embryo for the seeds of primary, secondary, tertiary umbels (1518.9,1443.3, and 816.8 µm) respectively in compare with the shortest embryos that found in B 0 C 2 .The results of the second order interaction between calcium and trehalose (Table 3B) shows the consistent differences between treatments, T 2 C 1 treatment increased embryo length in primary, secondary, tertiary umbels (1596.3,1504, and 833.5 µm) respectively compared to the lowest percentages in T 0 C 2 .The results of 3B table also show that the endosperm length in all umbels order didn't reach to significant level.4B.Table 3 A. Germination speed (G P )(%) and germination percent (G S ) (%) of primary, secondary, tertiary umbels of carrot seeds after treatment for two ways interaction The results of Table (4 B) indicate that there were significant differences in second order interaction between the aqueous extract of barley sprouts and trihalose.(B 1 T 2 ) produced the highest percentages of GP and GS for the seeds of primary (83.89, 88.89%), secondary (80.89, 86.78%), tertiary umbels (G S only) (66.33,) respectively.With reference to the interaction between aqueous germinated barley grains extract and calcium (Table 4B); B 1 C 1 shows the highest percent of GP and GS for the seeds of primary (83.89, 88.89%), secondary (80.89, 86.78%), tertiary umbels (G S only) (66.33) respectively.The results of the second order interaction between calcium and trehalose (Table 4B ) that there is a significant effect of the third order interaction treatments in the percentages of GP and GS for the seeds of all umbels orders.B 1 T 2 C 1 treatment produced the highest percentages (92, 97%); (90.67, 94.67%); (75.68, 86.33%) respectively, compared to the lowest percentages in B 0 T 0 C 2 .

Figure 2. variable embryos length in second
order umbels as affected by study treatments Exogenous application of sprouted barley aqueous extract, trehalose, and calcium was efficiently utilized by increasing embryo length.The elevating impact of barley sprouts aqueous extract, trehalose, and somehow calcium, and in the process of embryogenesis and fruit development, which led to the growth of the embryo and its increase in its length compared to the endosperm, achieving several treatments first-and second-order, the required maturity rate (length of the embryo = twothirds of the length of the endosperm), and overcoming the problem of ruidementary or immature embryos (figure 2) (25).and then obtain regular germination and emergence, as several studies have confirmed the work of the trehalose precursor in embryos maturation in the Arabidopsis plant through its contribution to completing the embryogenesis stages of the process to the fullest extent (9), therefore Trehalose metabolism is important in regulating food reserves, as T 6 P is essential for utilization of carbohydrates (22).Moreover; Calcium regulates the growth of developing fruit in carrots after fertilization) due to its direct action in forming cell walls and maintaining the structure and permeability of cell membranes.These results agree with (18,19).the effect of sprouted barley aqueous extract is came from the bioavailable nutrients that had (Table 2), which had a role in fruit development and seeds, such as nitrogen, phosphorus, and potassium (7,14)

Figure 1 .
Figure 1.The longitudinal section of carrot seed from the first third of the seed The collected data analyzed using analyses of variance and the means were compared according to L.S.D. test under 5% probability.RESULTS AND DISCUSSION 1.Embryo and endosperm length (µm) of primary, secondary, tertiary umbels of carrot seeds: The results of the statistical analysis of the individual factors show the significant superiority of B 1 in producing the longest embryo in primary secondary, and tertiary umbels (1335.9,1255.3,704.3 µm) respectively.In compare with the shortest embryos which found in B 0 (Table3A).Plants that received foliar trehalose application (75 mmol/l -1 ) exhibited longer embryos in all