Plant Cell Reports

Plant Cell Reports (1986) 5 : 381-384

© Springer-Verlag 1986

Shoot and root organogenesis of Camellia sasanqua Kenneth C. Torres and Jacqueline A. Carlisi Department of Horticulture, 137 Julian C. Miller Hall, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA Received March 25, 1986 / Revised version received August 13, 1986 - Communicated by G. C. Phillips

ABSTRACT

MATERIALS AND METHODS

In v i t r o - d e r i v e d shoot t i p s , (10 mm) taken from p r i o r yc----c-uTtures of Camellia s a s a ~ L . , were evaluated for organogenesis when cultured on a half-strength MS medium supplemented with various concentrations of NAA, IBA, BA and GA3. Maximum shoot p r o l i f e r a t i o n and growth f o r juvenile and mature tissue was obtained when 0.54 uM NAA, 8.8 ~M BA plus 14.4 to 28.9 uM GAq was added to the culture media, with a pH between 4.5 and 5.0. In v i t r o - d e r i v e d shoots (20 mm) from mature C. ~ 'Day Dream' and juvenile C. sasanqu~ i n i t i a t e d roots in v i t r o ~ f t e r Immersion in 2.5 mM IBA for 30 min.---Sl-~-£Tpercent of the mature shoots and 90% of the juvenile shoots i n i t i a t e d roots within 3 weeks of treatment with IBA.

Herbaceous softwood stem segments, (100 mm) were excised from 6- to 8-month-old (juvenile) plants of C. sasanqua and from approximately 10 y r - o l d (maTure--~ plants of C. s ~ 'Day Dream'. Stem segments used as primary explants were taken from the present years growth. Explants taken in the spring were surface s t e r i l i z e d with 1.05% sodium hypochlorite plus 0.1% Tween 20 for 5 min and then rinsed 3 times with s t e r i l e d i s t i l l e d water. Due to increased contamination during the summer months, tissue obtained in the summer was pretreated with 95% ethyl alcohol for I min, surface s t e r i l i z e d for I0 min with the above s t e r i l a n t solution and then rinsed three times in double d i s t i l l e d water. Leaves were removed from the stem a f t e r s t e r i l i z a t i o n and the stem segments were cut into I0 mm nodal sections. One nodal section was placed into each 20 x 150 mm culture tube. Juvenile tissue was maintained on I0 ml of a culture medium containing 4.4 uM BA plus 0.54 uM NAA and mature tissue was maintained on I0 ml of a culture medium containing 4.4 uM BA alone ( C a r l i s i , 1985). Previous work indicated that the percentage of mature nodal sections established in v i t r o could be enhanced by placing the cultures the dark f o r the f i r s t 2 weeks followed by i~cu~ation under a 16 h photoperiod with 100 ~Esec~m-~ of l i g h t ( C a r l i s i , 1985). One or more shoots p r o l i f e r a t e d from the nodes which were then subcultured onto an identical medium for further p r o l i f e r a t i o n and subsequently used as the explants for a l l other experiments. Preliminary experiments indicated the best basal medium to be half-strength MS salts containing 8 g / l i t e r Difco-Bacto agar, 0.001 mM thiamine-HCl, 0.50 mM myo-inositol and 87.6 mM sucrose ( C a r l i s i , 1985). All growth regulators were added p r i o r to autoclaving except f o r GA3 and IBA which were f i l t e r s t e r i l i z e d through a 0.2 um f i l t e r and added a f t e r s t e r i l i z a t i o n . The pH of a l l media, except for the pH study, was adjusted to 5.0 with 1.0 N HCI ~r 1.0 N NaOH prior to autoclaving at 1 kg/cm for 15 min at 121°C. Media pH did not change (± 0.1 pH unit) during autoclaving. Medium f o r the pH study was adjusted to 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0. All cultures were maintained at 25°C ± 2°C. Data f o r a l l experiments were collected a f t e r an 8 week

ABBREVIATIONS:

MS, Murashige and Skoog (1962); IBA, IH-indole-3-butanoic acid; BA, N-(phenyl-methyl)-IH-purine6-amine: GA~, g i b b e r e l l i c acid; k i n e t i n , N-(~-furanyl-methyl)IH-purine-6-amine; NAA, l-naphthaleneacetic acid; L, Linear; Q, Quadratic.

INTRODUCTION Camellias are a popular flowering woody ornamental. Some species are widely adapted to many climates and are tolerant of shade and adverse environmental conditions. Commercially, Camellia japonica and C. sasanqua are propagated -~-#-utt-ings. Bennett ~ n d ~ r t (1982) reported adventitious shoot development when primary cotyledonary callus of C. was cultured on a low s a l t medium co-ntaining 2.2 to 22.0 uM BAo Samartin et a l . (1984) reported shoot production from shoo~t-Tp explants of 3- to 4-month-old seedlings of C. ~ o n a modified MS medium containing 4.4 ~ M BA. Successful rooting was achieved by dipping the shoots in 2.5 mM IBA f o r 20 min then transferring onto a MS medium without hormones. In contrast, a micropropagation system f o r C. sasanqua has not been reported to date. The purpose of t h i s research is to define the in v i t r o m u l t i p l i c a t i o n and rooting requirements fo-g~, sasanqua.

Offprint requests to: K. C. Torres

382 culture period. Each experiment was i n i t i a t e d with 10 replicates per treatment. A 5 x 5 f a c t o r i a l experiment with NAA (0.0, 0.54, 2.7, 5.4 and 10.8 uM) and BA (0.0, 0.44, 2.2, 4.4, 8.8 ~M) was used to evaluate the number of new shoots and shoot length. Shoot lengths greater than 20 mm were considered to be essential from preliminary work f o r e f f i c i e n t rooting. A 2 x 6 f a c t o r i a l experiment with BA (4.4, I I . 0 pM) and GA. (0.0, 0.3, 1.4, 2.9, 14.4 and 28.8 ~M) was used t~ evaluate shoot length and number. Root i n i t i a t i o n from in vitro-derived shoots (20 mm) was evaluated by~ipp--p-fng the base (5-7 mm) into 2.6 mM NAA or 2.5 mM IBA for 10, 20, or 30 min. The shoots were placed onto the basic culture medium for 8 weeks. Both IBA and NAA were evaluated for root induction from mature-derived shoots while only IBA was evaluated f o r root induction from juvenile-derived shoots. The number of roots produced per shoot and percent of cultures i n i t i a t i n g roots were recorded. Experimental results were analyzed using the least squares analysis of variance program. Means were separated and significance determined using Duncan's multiple range t e s t . Orthogonal polynomial comparisons were used to test for significance between hormone concentrations when more than 2 concentrations were used. Chi square tests were u t i l i z e d to test for significance among percentages. Table 1.

RESULTS AND DISCUSSION NAA and BA effects. The interaction between NAA x BA on shoot production was non-significant; however, the main e f f e c t was s i g n i f i c a n t (P < b.Ol). As BA concentration increased, shoot number increased (Figure 1). Conversely, shoot number decreased with increasing NAA levels (Figure 1). A s i g n i f i c a n t interaction existed between NAA and BA for shoot length (Table i ) . Maximum mean shoot length of 33 mm was observed when 2.2 ~M BA and 0.54 pM NAA was added to the culture medium (Table 1). Shoot elongation was depressed as BA concentrations exceeded 4.4 ~M (Table I , Figure 1). The percent of cultures forming shoots greater than 20 mm was highest when 0.44 ~M or 2.2 uM BA was combined with 0.54 ~M NAA (Table I ) . The percentage of shoots greater than 20 mm generally was inhibited by higher concentrations of both NAA and BA (Table I ) . Samartin et a l . (1984) reported optimal shoot p r o l i f e r a t i o ~ - - f ~ m j u v e n i l e _C. japonica with 4.4 ~M BA. They obtained 2.6 shoots per explant with the height of the t a l l e s t shoot equal to 30 mm. A greater number of shoots, however, was obtained with 8.8 pM BA and 4.6 ~M k i n e t i n , but the length of the t a l l e s t shoot was only 17 mm (Samartin et a l . , 1984). Our results demonstrate that as m~y as 6.0 shoots per explant could be obtained when 8.8 uM BA was used alone or combined with 0.54 uM NAA.

The effects of NAA and BA on the elongation of in vitro-derived shoots from juvenile ~. sasanqua. BA (~M)

NAA (~M) 0.0 0.54 2.7 5.4 10.8

0.0 21.1 21.2 26.3 15.0 10.6 18.9

0.0 0.54 2.7 5.4 10.8

45 50 62 25 0 36

0.44

2.2

- - - average shoot length (mm)z - - 18.3 26.9 27.1 33.3 21.4 24.1 16.7 21.8 I0.0 20.0 18.0

25.7

- - - percent of shoots > 20 mmy - - 34 67 71 64 43 41 34 36 0 50 36

52

4.4

8.8

15.3 15.7 20.0 20.0 17.2

11.7 10.7 9.7 10.8 14.4

16.9

11.3

27 30 44 36 44

8 10 15 13 21

36

14

16.1 16.4 16.5 14.8 14.6

36 46 41 30 21

ZBAQ, highly s i g n i f i c a n t (P < 0.001); BAL * NAAQ, s i g n i f i c a n t (P < 0.01) Ychi square test used f o r percentages BA, highly s i g n i f i c a n t (P < 0.001); BA * NAA, highly s i g n i f i c a n t (P < 0.001) GA3 and BA effects. When averaged over a l l GA3 concentrations, 11.0 pM BA produced s i g n i f i c a n t l y more shoots per explant (5.9 compared to 3.6) than 4.4 ~M BA. Shoot length, averaged over a l l GA~ concentrations, was s i g n i f i c a n t l y greater (22.~ to 16.3 mm) with 4.4 uM BA than 11.0 ~M BA. Although shoot production was not s i g n i f i c a n t l y influenced by GA3 l e v e l s , the highest number of shoots produced per explant was observed at 28.8 uM GA~ plus 11.0 ~M BA. Shoot length increased with ~ncreasing GA3 concentrations (Figure 2). Low concentrations of GA3 were more e f f e c t i v e in increasing shoot length at 4.4 pM BA than at I i . 0 ~M BA. Concentrations of GA3 greater than 14.4 uM were required to promote shoot elongation greater than 20 mm at 11.0~M BA.

The use of GA~ in shoot t i p cultures has been reported for mic~opropagation of apple (Jones et a l . , 1977; Lane, 1978), blackberry (Broome and-~mmerman, 1978) and other species (George and Sherrington, 1984). Lane (1978) reported that the omission of GA3 resulted in apple shoots which had shortened internodes and deformed leaves. Shoot elongation in Phlox was also dependent upon the presence of G A ~ h n a b e l r a u c h and Sink, 1979). Our results wi~h C. sasanqua demonstrate that optimal shoot proTiferatlon and elongation can be simultaneously obtained by combining 11 ~M BA with 28.8 ~M GA3 (Figure 2). Further enhancement of shoot p r o l i f e r a t i o n and elongation might be achieved by e i t h e r greater concentrations of BA and/or GA3.

383

.60 E E

.BA

5

o (,~

A 5%_

k~

E ~

Z

,40~

4

o 0 .c~ o~

o

~0~

3 >

< 2

..........

• Average Shoot Length • Percent Shoots at 4.4uM BA • Percent Shoots at 11.0uM BA

20~ i~ D_

1o

I

0.0

|

2.0

.

|

4.0

I

6.0

I

8.0

I

10.0

,,

,

0.0

Growth Regulator [uM)

Figure I ,

The e f f e c t s of BA and NAA on the product i o n of shoots (mean ± S.E.) from nodal section explants of j u v e n i l e C. sasanqua. BAQ (P < 0.001) NAAL (P < O.OT)'

pH e f f e c t s . The pH of the culture media influenced shoot p r o l i f e r a t i o n and shoot length for j u v e n i l e and mature tissue (Figure 3), Shoot production from immature and mature tissue increased as media pH increased from 3.5 to 5.0 (Figure 3a). A reduction in shoot production was observed at a pH of 6.0. The pH had a greater e f f e c t on shoot p r o l i f e r a t i o n from j u v e n i l e tissue than from mature tissue (Ffgure 3a). Greatest shoot elongation occurred at pH 4.5 (Figure 3b). The regulation of morphogenesis in several species has been shown to be influenced by the i n i t i a l pH of the c u l t u r e medium (Sharma et a l . , 1981; Takayma and Misawa, 1979; Venverloo~--1~-~8). Sharma et a l . (1981) noted that rooting of in v i t r o - d e r i v e d shoots of Bou~ainvillea was e-~-anced when the pH of the c u l t u r e medium was adjusted i n i t i a l l y to 4,5, Venverloo (1978) noted that a culture media pH of 5.0 to 6.3 resulted in the best rooting of Nautilocal~x. Bulblet formation in L i l i u m auratum was' best in c u l t u r e medium adjusted to pH-~.O to 7.0 (Takayama and Misawa, 1979), Results from our research indicate that best shoot p r o l i f e r a t i o n occurred when the pH of the media was adjusted to 5.0 p r i o r to sterilization. IBA and NAA e f f e c t s . Rooting of in v i t r o derived shoots of j u v e n i l e C. sasanqua was s i g n i f i c a n t l y influenced by the immersion time in 2.5 mM IBA (Table 2). A two-fold increase in the number of roots resulted when immersion time

7.2

14.4

21.6

28.8

GA 3 (uM]

Figure 2,

The effects of various concentrations of GA3 on the average shoot length and the percentage of shoots greater than 20 mm at 4.4 and 11,0 ~M BA produced from nodal sections of j u v e n i l e C, sasanqua in v i t r o . GA3L (P < 0.~01)

was increased from 10 to 30 min, Ninety percent of the cultures i n i t i a t e d roots when immersed f o r 30 min while only 50% of the cultures produced roots at ]0 min in IBA (Table 2). In contrast, rooting of in v i t r o - d e r i v e d shoots of mature C, sasanqua 'Day Dream' was not affected by the immersion time in e i t h e r 2.6 mM NAA or 2.SmM IBA. Auxin source did, however, influence the number of roots produced per ~. s,asanqua 'Day Dream' shoot. IBA produced an average of 1,9 roots per shoot whereas NAA produced less than 0.3 roots per shoot. Also, a higher percentage (59% compared to 18%) of the shoots treated with IBA i n i t i a t e d roots than those treated with NAA, Samartin et a l , (1984) indicate that rooting of j u v e n i l e ~. japonica is best when in v i t r o derived shoots are dipped in a 2.5 mM--i~B~solution f o r 20 min. A decrease in the percentage of shoots forming roots occurred when the shoots were immersed in IBA for 30 min. In contrast, our results with C. sa~anqu,a indicated that a higher percentage o f - r o o t i n g as well as a larger number of roots were obtained when the cuttings were immersed f o r 30 min in IBA. In conclusion our results indicate that -C. sa~can be e f f i c i e n t l y propagated through ~-~ssue c u l t u r e . Growth regulators and media pH influence shoot p r o l i f e r a t i o n as well as shoot elongation. Furthermore, the effects of growth regulators and media pH on shoot p r o l i f e r a t i o n and elongation d i f f e r s f o r j u v e n i l e and mature tissue.

384 Table 2.

The e f f e c t of IBA and immersion time on the rooting of in v i t r o - d e r i v e d shoots of juvenile ~. sasanqua.

Immersion Time in IBA (min)

Mean Root Number

Percent of Cultures With Roots

10

2.1 bz

55y

20

2.6 ab

60

30

4.3 a

90

ZMeans separation by Duncan's multiple range test at 5% level Ychi square test used for percentages immersion time, s i g n i f i c a n t (P < 0.05)

A

J u v e n i l e [] Mature O

Juvenile Mature

& ~ -25

5.0