PHILIPPINE COCONUT AUTHORITY 2004 RESEARCH ACCOMPLISHMENT HIGHLIGHTS

 
 
 

BIOTECHNOLOGY PROGRAM (TISSUE CULTURE DIVISION )

 

 

Division Chief III Erlinda P Rillo, Scientist IV, Plant Pathology (MS)

Sr. Science Research Specialist Cristeta A. Cueto, Plant Pathology (MS)

Science Research Specialist II Osmundo D. Orense , Plant Biotechnology (MS)

Science Research Specialist II Maria Buena B. Areza-Ubaldo, Plant Science (MS)

Science Research Assistant Leo Alexi Imperial (BS)

 

 

Coconut tissue culture for clonal propagation and safe germplasm exchange (PCA-ACIAR)

 

Verification study on the effect of exogenous application of ABA on ex vitro survival of embryo cultured seedlings

Embryo cultured Laguna seedlings (cultures established in 2003) were treated with 0, 10, 20, 30, 40, and 50 µM abscissic acid ( ABA ) one month prior to potting-out. Treatments were replicated 2 times with 14 samples per replicate. The survival of the seedlings was assessed 3 months after potting-out (2004). Data were analyzed using the Analysis of Variance (ANOVA) and means were compared using Duncan 's Multiple Range Test (DMRT).

Table 1 shows that addition of ABA one month prior to potting out had no significant effect on the ex vitro survival of 5 month-old embryo cultured LAGT seedlings. This suggests that there is no need to incorporate ABA in the embryo culture medium thereby reducing production cost.

Table 1. Ex vitro survival of embryo cultured seedlings with different levels of ABA three months after potting-out.

 

ABA Treatment (µM)

Percent Survival

0

96.50 ab

10

90.50 ab

20

100.00 a

30

87.00 ab

40

84.50 ab

50

96.50 b

Mean

92.50

Pr>F, Treatment

0.164

Statistical Significance

ns

Coefficient of Variation (CV)

5.86

ns Treatment effect is not significant; Treatment means with the same letter(s) are not significantly different from each other at 5% level.

Effect of different levels of sugar (applied during the last 2 passages) on ex vitro survival of seedlings

The effect of different levels of sugar during the last 2 passages prior to potting-out of embryo cultured seedlings was compared in terms of survival of the seedlings in the soil. The experimental materials were embryo cultured seedlings cv. Malayan Yellow Dwarf (MYD) which were initiated using the improved embryo culture protocol. The seedlings were exposed to different sugar levels (T1-25 g/L, T2-35 g/L, T3-45 g/L as control) during the 3 rd and 4 th month after initial culture before potting-out. Treatments were replicated two times with 18 samples per replicate. Ex vitro survival of the seedlings was assessed 3 months after potting-out. Data were analyzed using the ANOVA and means were compared using DMRT.

Table 2 shows that there were no significant differences in the survival of the embryo cultured MYD seedlings in the soil and the number of expanded leaves produced 3 months after potting-out among the sugar level treatments. Results indicated that the initial amount of sugar (60 g/L) in the growth medium may be reduced to 25, 35 or 45 g/L during the last two passages (3 rd & 4 th month after initial culture) of embryo culture without any detrimental effect on growth and survival ex-vitro .

Table 2. Ex vitro survival and number of expanded leaves produced 3 months after potting-out of seedlings.

 

Sugar Treatment

(g/L)

Ex vitro survival

(%)

No. of expanded leaves produced

0

1

2

3

4

5

25

88.50 a

3.00 a

0 a

3.00 a

31.50 a

39.50 a

23.00 a

35

88.00 a

3.00 a

0 a

3.00 a

20.50 a

41.00 a

32.00 a

45

80.00 a

0 a

3.00 a

14.00 a

28.50 a

34.50 a

20.50 a

Mean

85.50

2

1

6.67

26.83

38.33

25.17

Pr>F, Treatment

0.604

0.750

0.500

0.648

0.789

0.864

0.705

Statistical Significance

ns

Ns

ns

ns

ns

ns

ns

CV

9.75

212.13

244.95

182.89

58.05

31.65

52.59

ns Treatment effect is not significant; Treatment means in the same column with the same letter(s) are not significantly different from each other at 5% level.

Effect of haustorium removal on growth of coconut embryos in vitro and seedling survival ex vitro

The necessity for long incubation period of seedlings in vitro could be attributed to their slow growth. It is perceived that the presence of haustorium influences the slow development of the seedlings in vitro . Besides, the haustorium sometimes tends to grow faster than the seedling filling tightly the circumference of the test tube hindering the easy transfer of seedlings and, often, injuring the seedlings during subculture. Removal of the haustorium will facilitate easy transferring of seedlings from one test tube to another during subculture and may promote better development of seedlings in vitro .

It is believed, however, that the haustorium can store nutrients which may be utilized by the seedlings during transition from in vitro to ex vitro condition. If this is so, the haustorium can then be allowed to grow to a certain limit, which will not restrict the seedling growth inside the test tube. In this way, haustorium-stored nutrient would be available for the plant while adapting to ex vitro condition during soil establishment.

The experiment was conducted to determine the effect of removing the haustorium on in vitro growth and ex vitro survival of seedlings. The experiment used zygotic embryos from Laguna Tall (LAGT) and Malayan Yellow Dwarf (MYD). The treatments were as follows:

T1 Haustorium cut immediately after germination

T2 Haustorium cut 1 month after germination

T3 Haustorium intact during the entire culture duration

No difference in shoot length and seedling fresh weight was observed in LAGT and MYD 5 months after culture with or without the removal of haustoriim (Table 3). This showed that the presence or absence of the haustorium did not affect seedling development in vitro . The presence of enlarged haustorium in the cultures, however, made transfer of seedlings during subculture more difficult and resulted to seedling injury.

Table 3. Effect of removing the haustorium on in vitro growth performance and ex-vitro survival of LAGT and MYD seedlings after 5 months of in vitro culture.

 

 

Treatment

Growth Parameter

Ex vitro Survival

(%)

Ave. Shoot Length (cm)

Ave. Fresh Weight (g)

LAGT

 

Haustorium cut immediately after germination

12.98 ns

7.22 ns

 

Haustorium cut 1 mo after germination

12.86 ns

7.42 ns

 

Haustorium Intact

13.12 ns

6.94 ns

 

MYD

 

Haustorium cut immediately after germination

12.23 ns

5.86 ns

100 ns

Haustorium cut 1 mo after germination

12.54 ns

6.20 ns

100 ns

Haustorium Intact

12.14 ns

5.88 ns

100 ns

ns Treatment effect is not significant

There was also no difference in the ex vitro survival rates of MYD seedlings 5 months after culture. On the other hand, ex vitro survival rates for LAGT could not be ascertained due to contamination of cultures in vitro . These results implied that cutting the haustoria of developing seedlings immediately after or 1 month after germination had no effect on ex vitro survival of in vitro cultured seedlings. The same findings were observed in the culture of Makapuno embryos (Data not shown).

The very high survival rates observed in MYD seedlings could be partially attributed to the use of a humidity tent in the soil establishment part of the experiment (Figure 1). The use of this humidity tent resulted in less disturbance of seedlings during inspection and provided a bigger headspace of humidity for the developing seedling .

It was also observed that even after 5 months of in vitro culture, the haustoria of many developing seedlings were not enlarged and sometimes even inconspicuous. The theory that it can store nutrients for seedling development during ex vitro establishment, therefore, may not be true.


In vitro culture planting protocol to hasten hardening of plantlets towards early soil transplantation

The experiment tried to determine any difference in nutrient uptake of in vitro grown seedlings if only roots and the tip of the haustorium or if roots, haustorium and part of the stem are in contact with the nutrient medium. It is believed that hardening of seedlings in vitro would be faster with nutrient uptake only through the roots and haustorium. Subsequently, the roots will be trained to function fully in vitro and, hence, contribute to seedling survival ex vitro . The experiment used zygotic embryos of Laguna Tall (LAGT) and Malayan Yellow Dwarf (MYD).

The treatments were as follows:

T1 Only the roots and tip of the haustorium are submerged

in the nutrient medium

T2 The roots, haustorium and part of the stem are submerged

in the liquid nutrient medium (control)

For T1, the cultures were supported inside the test tube with sterilized pieces of polyurethane stuffing material (Figure 3). Growth of LAGT and MYD seedlings were generally slower when only the roots and tip of the haustorium are submerged in the nutrient medium as compared to seedlings with the roots, haustorium and part of the stem submerged in the medium (Table 4). This indicated that absorption of nutrients by the seedlings is increased when petioles and stems are also in contact with the nutrient medium.

However, ex-vitro survival of seedlings were significantly higher in seedlings where only the roots and tip of haustorium were in contact with the medium during in vitro culture. The higher ex vitro survival rates could be attributed to the better development of functional roots during in vitro culture.

Figure 3. Four month-old MYD seedlings planted as usual (T2) and oriented in such a way that only the roots and tips of haustoria are in contact with the liquid medium (T1)


 

Figure 3. Four month-old MYD seedlings planted as usual (T2) and oriented in such a way that only the roots and tips of haustoria are in contact with the liquid medium (T1)

 

UPDATES

NOVEMBER 2005

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