Candidate Biol. Sci. Malyarovskaya V.I.

The State Research Institution All–Russian Scientific and Research Institute of Floriculture and Subtropical Crops of the Russian Academy of Agricultural Sciences, c. Sochi, Russia, e-mail: lab-bfbr@vniisubtrop.ru

SOME CHARACTERISTICS OF CULTIVATING ANTHERS FROM DIFFERENT CULTIVARS OF DIANTHUS CARYOPHYLLUS IN VITRO

The paper presents the study results on cultivating anthers from different cultivars of Dianthus caryophyllus in vitro. It is revealed that its genotype has the greatest impact on the efficiency of anther culture in vitro. According to the research of Dianthus caryophyllus different genotypes, cultivars with high androgenic potential were allocated by responsiveness to in vitro conditions. These include the following cultivars: Cristobal, Carnaval, Amapola, Salome and Lena. It has been established that the ability to induce proliferation processes, intensity of their transmission as well as the regeneration is dependent on the composition and concentration of phytoregulators.

Key words: Dianthus caryophyllus, anther culture, callus formation, embryogenesis, embryonic cell complexes, regenerated plants, cytological control, diploid and haploid cells.

Introduction

One of the most common ways to get doubled haploids is a method of induction of androgenesis by cultivating the selected anthers. This method has been successfully applied for many crops, particularly barley [1, 2], wheat and triticale [3,4], maize [5], rapeseed, mustard [6], flax [7] and others.

However, as we know, it is difficult to produce haploid plants applying the methods of androgenesis because of a few growths (calluses and embryoid-like structures (ELS)) and a low percentage of plant regeneration [8,9,10].

Numerous literature on the theoretical and methodological aspects of cultivating anthers of different crops in vitro indicate that genotypic dependence of haploid-productive process is the most serious obstacle to the introduction of haploid technologies into the selection [11,12,13]. In addition, recommendations on the use of only responsive forms in anthers culture [14] and on the allocation of donors having high regenerative capacity became widespread [15].

In connection with this, the aim of the research was to study the genotypic dependence of Dianthus caryophyllus on the ability to form androgenic structures in anther culture in vitro.

Research technique

The research was conducted in 1986-1990 yy. on the basis of Biotechnological and Selection Departments in the Institute of Floriculture and Subtropical Crops. 22 different types of Dianthus caryophyllus were used as explants donors (anthers): Lena, Arthur Sim, William Sim, Flamingo, Jaguar, Razdolnenskaya, Plamya, Yubileinaya, Originalnaya, Palace, Cristobal, Vanessa, Le Re, Amapola, Mayela, Carnaval, Claire Jellow, Samantha, Salome, Sandra, Elsie White Sim and eight hybrids: ОG-75-3, ОG-87-1, ОG-150-6, 3К-84-1, 3К-130-3, 5К-56-1, 8G-18-3, 3C-93-2.

Preparation of plant material and its introduction to the tissue culture was carried out with the requirements of aseptic techniques and methods of planting into medium recommended by R.G. Butenko [16].

Before introducing anthers into in vitro culture, 72-hour pretreatment with low positive temperature (+5 °C) was performed. Immediately before planting the anthers into culture medium, there was carried out a cytological control over pollen developmental stages on temporary and pressure preparations which had been painted with acetoorsein or acetocarmine solution, and analyzed them under a microscope MBI-15. The anthers were isolated on a mononuclear pollen stage. New growths obtained from anther culture (callus) were investigated, using temporal cytological preparations painted with acetocarmine or hematoxylin, according to standard procedures [17]. Callus cells were photographed (photomicrography) and sketched, using a drawing apparatus (DA).

Carnation buds of about 1,0 cm were sterilized in ethanol (96 %) for 5-7 minutes (laminar box), then washed 2-3 times with distilled water. Anthers were then aseptically singled out from the buds and placed into Murashige and Skoog medium (1962) with different ratios and concentrations of growth regulators: Option 1 – kinetin – 0,1 mg/l, adenine – 0,5 mg/l; Option 2 - 6 BAP – 1 mg/l, NAA – 0,3 mg/l IAA – 0,5 mg/l; Option 3 – adenine – 2 mg/l, NAA – 0,5 mg/l, 2,4-D – 3,0 mg/l, IAA – 0,5 mg/l; Option 4 - 6 BAP – 0,5 mg/l, zeatin – 0,1 mg/l; Option 5 – kinetin 2,0 mg/l, 2,4-D – 2,0 mg/l.

Anthers were cultivated in a nutrient medium at a temperature of 24 °C, air relative humidity was 65-70 %, firstly during 7 days in the dark, and subsequently in a photoperiod of 16/8 with to 1500 lux illumination. In 1,5-2 months the anthers with new formations (callus, embryonic cell complexes (ECC), rhizogenesis) were counted.

Results and discussion

As a result of the studies, it was found that the development of anthers from different cultivars of Dianthus caryophyllus in vitro mainly passes through callus forming, i.e. by a way of indirect androgenesis. At the same time there was not recorded a more desirable way of direct embryogenesis to form embryoids on anthers.

Anthers cultivation in nutrient media differing in composition and concentration of growth regulators showed that the pace and nature of development of new growths in different carnation cultivars were not the same. Thus, the high frequency of callus formation (25,7%) was observed in a cultivar Lena, in the medium at a concentration of phytohormones: 6 BAP – 1 mg/l, NAA – 0,3 mg/l, IAA – 0,5 mg/l (Table 1). However, the induction of embryonic cell complexes (2,1 %) was observed on another option of the nutrient medium. The overall incidence of new growths (47,2 %) in this cultivar was higher than that of the other carnation cultivars. For the cultivars from Sim group, such as: William Sim, Arthur Sim, White Sim and Flamingo, an active callus formation was observed in the nutrient medium containing adenine - 2 mg/l, NAA – 0,5 mg/l, 2,4-D – 3,0 mg/l, IAA – 0,5 mg/l (15,6 %, 18,9 %, 11,5 % and 13,9 %, respectively). Formation of embryonic cell complexes in anther culture was more active in the cultivar White Sim (2,7 %) on the second option of the culture medium. The third group with a high frequency of callus formation activity included French carnation cultivars: Cristobal, Carnaval, Amapola, Salome and Vanessa. The best ratio of phytohormones for them was the option with BAP 6 – 0,5 mg/l, zeatin – 0,1 mg/l.

Table 1. Effect of growth regulators on the initiation of new growths in anther culture of Dianthus caryophyllus.

Genotype	Induction of growths, %										Total, %
	Option- 1		Option - 2		Option -3		Option - 4		Option -5
	Cal-lus	ECC	Cal-lus	ECC	Cal-lus	ECC	Cal-lus	ECC	Cal-lus	ECC
Lena	4,1	0,0	25,7	1,8	0,9	2,1	3,8	0,0	8,1	0,7	47,2
William Sim	1,9	0,1	5,9	3,1	15,6	0,4	3,2	0,0	5,3	0,0	35,5
Arthur Sim	4,2	0,0	7,1	0,0	18,9	0,2	2,2	0,0	3,1	0,0	35,7
White Sim	0,9	0,0	8,3	2,7	11,5	1,8	0,9	0,0	0,0	0,0	26,1
Flamingo	3,4	0,0	6,7	1,2	13,9	0,0	3,1	0,0	1,4	0,1	29,8
Cristobal	6,9	0,0	3,8	1,1	4,8	0,0	20,1	0,0	2,7	0,0	39,4
Carnaval	7,3	0,0	4,9	0,1	2,4	0,0	17,9	0,0	0,9	0,0	33,5
Amapola	8,2	0,0	6,5	0,0	3,9	0,0	18,5	0,0	0,0	0,0	37,1
Salome	2,3	0,0	3,5	0,0	1,1	0,0	13,1	0,0	0,0	0,0	20,0
Vanessa	0,0	0,0	4,0	0,5	4,1	0,0	10,9	0,0	0,0	0,0	19,5
3К-84-1	2,4	0,0	4,9	0,1	1,1	0,0	5,9	0,0	3,0	0,0	17,4

New growths with low frequency were observed in anther culture of the hybrid material. From the eight studied hybrids, only hybrid 3K-84-1 had callus and embryogenesis.

More than half of the calluses (58%) had a dense white texture, they quickly turned green in the light. These calluses were induced from anther culture on the second option of the culture medium for the cultivar Lena, and on the fourth option for the cultivars: Cristobal, Carnaval, Amapola, Salome and Vanessa. These calluses had morphogenic zones. There were also non-morphogenic calluses - loose, watery, colorless or yellow. The cultivars William Sim, Arthur Sim, White Sim, Flamingo and almost all hybrids had from 27 to 63 per cent of such calluses. Conducted cytological control over the processes of embryogenesis in callus culture of Dianthus caryophyllus isolated anthers showed that callus tissues consist of various cells in size and shape (Fig. 1. a-i).

а	b	c
d	e	f
g	h	i

Fig.1. Callus cells of different shapes and sizes induced from Dianthus caryophyllus

anthers (by Ryzhkova L.V., Skipina K.P., 1988-1989 yy.): a. - procambial cells of callus in Carnaval cultivar (increased in 90x10x7); b. -1. callus cells of Carnaval cultivar, 2. procambial cells, 3. pollen tubes (increased in 16x10x1,6); c. - 1. callus cells of Carnaval cultivar 2. - coil vessels, 3. - meristematic cells (increased in 16x10x1,6); d.e. - callus cells of Cristobal cultivar, 1., 2 - core (increased in 10x10x1,6); f. - 1. and 2. callus and procambial cell of Cristobal cultivar ( increased in 40x10x1,6); g. - diploid callus cell of Carnaval cultivar, 1. - callus cells, 2. - core 3. - chromosomes ( increased in 16х10х1,6 и 60х10х1,6); h. - diploid and haploid callus cells of Cristobal, 1. core and 2. chromosomes ( increased in 60x10x1,6); i. - 1. procambial haploid callus cell of Carnaval cultivar, 2. - core, 3. - chromosomes.

Except for large callus cells with loose cytoplasm, in callus tissue derived from Carnaval and Critobal anthers there were found groups of procambial cells having substantially rectangular shape and pollen tubes, which were formed during the germination of pollen that continued to develop in a nutrient medium until full maturity (Fig. 1 b, d, e). Meristematic hotbeds were also observed with small, rounded cells with a dense cytoplasm and sieve tubes (Fig. 1c).

Counting of chromosomes in callus cells of these varieties showed that larger cells had a diploid number of chromosomes (2n=30), while smaller cells had a haploid set of chromosomes (n=15) (Fig.1 g, h, i).

With the aim to induce morphogenic structures in the obtained androgen calluses of D. caryophyllus they were passaged to special, so-called regenerative medias with higher cytokinin content, compared to auxin,: 6-BAP- 1,0 mg/l, zeatin- 0,5 mg/l, NAA - 0,1 mg/l. After 21 days of cultivation of callus, induced from carnation anther of Lena cultivar, we could observe the morphogenic structures from which we subsequently obtained regenerated plants. Conducted cytological analysis of the regenerated plants showed that they had a diploid set of chromosomes (2n = 30).

Conclusions

It is revealed that genotype has the greatest impact on the efficiency of anthers culture in vitro. According to the research of different Dianthus caryophyllus genotypes on responsiveness to in vitro cultivation conditions, cultivars with high androgenic potential were recorded. They include the following cultivars: Cristobal, Carnaval, Amapola, Salome and Lena. It has been established that the ability to induce proliferation processes, the intensity of their transmission and then the regeneration also depend on the composition and concentration of phytoregulators.

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